ORIGINAL RESEARCH

published: 07 November 2019

doi: 10.3389/fmicb.2019.02462

Comparative Evaluation of Different

Sanitizers Against Listeria
monocytogenes Biofilms on Major
Food-Contact Surfaces
Zi Hua1†, Ahmed Mahmoud Korany1,2†, Saadia Helmy El-Shinawy2 and Mei-Jun Zhu1*
1
School of Food Science, Washington State University, Pullman, WA, United States, 2Food Hygiene and Control Department,
Faculty of Veterinary Medicine, Beni-Suef University, Beni Suef, Egypt

Contaminated food-contact surfaces are recognized as the primary reason for recent L.
monocytogenes outbreaks in caramel apples and cantaloupes, highlighting the significance
of cleaning and sanitizing food-contact surfaces to ensure microbial safety of fresh
Edited by:
produce. This study evaluated efficacies of four commonly used chemical sanitizers at
Viduranga Y. Waisundara, practical concentrations against L. monocytogenes biofilms on major food-contact
Australian College of Business and surfaces including stainless steel, low-density polyethylene (LDPE), polyvinyl chloride
Technology, Sri Lanka
(PVC), polyester (PET), and rubber. In general, efficacies against L. monocytogenes biofilms
Reviewed by:
Kihwan Park, were enhanced by increasing concentrations of quaternary ammonium compound (QAC),
Chung-Ang University, chlorine, and chlorine dioxide, or extending treating time from 1 to 5 min. The 5-min
South Korea
Xuetong Fan,
treatments of 400 ppm QAC, 5.0 ppm chlorine dioxide, and 200 ppm chlorine reduced
United States Department of 3.0–3.7, 2.4–2.7, and 2.6–3.8 log10 CFU/coupon L. monocytogenes biofilms depending
Agriculture, United States on surfaces. Peroxyacetic acid (PAA) at 160 and 200 ppm showed similar antimicrobial
*Correspondence: efficacies against biofilms either at 1- or 5-min contact. The 5-min treatment of 200 ppm
Mei-Jun Zhu
[email protected] PAA caused 4.0–4.5 log10 CFU/coupon reduction of L. monocytogenes biofilms on tested
†
These authors have contributed surfaces. Surface material had more impact on the efficacies of QAC and chlorine, less
equally to this work influence on those of PAA and chlorine dioxide, while organic matter soiling impaired
sanitizer efficacies against L. monocytogenes biofilms independent of food-contact
Specialty section:
This article was submitted to surfaces. Data from this study provide practical guidance for effective disinfection of food-
Food Microbiology, contact surfaces in food processing/packing facilities.
a section of the journal
Frontiers in Microbiology Keywords: biofilm, L. monocytogenes, sanitizers, food-contact surfaces, organic matter, peroxyacetic acid
Received: 22 July 2019
Accepted: 14 October 2019
Published: 07 November 2019
INTRODUCTION
Citation:
Hua Z, Korany AM, El-Shinawy SH As a critical foodborne pathogen, Listeria monocytogenes causes approximately 1,600 cases of
and Zhu M-J (2019) Comparative infection and 260 cases of death annually in the United  States (Scallan et  al., 2011). It has
Evaluation of Different Sanitizers
been implicated in multi-state outbreaks on fresh produce including cantaloupes (CDC, 2012),
Against Listeria monocytogenes
Biofilms on Major
prepackaged caramel apples (CDC, 2015a), bean sprouts (CDC, 2015b), frozen vegetables
Food-Contact Surfaces. (CDC, 2016a), and packaged salads (CDC, 2016b) since 2011. Contaminated food-contact
Front. Microbiol. 10:2462. surfaces, packing lines, and environment are incriminated as the primary reasons linked to
doi: 10.3389/fmicb.2019.02462 L. monocytogenes outbreaks in fresh produce (McCollum et  al., 2013; Angelo et  al., 2017).

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Hua et al. Sanitizer Disinfection of L. monocytogenes

Therefore, it is vital to sanitize food-contact surfaces along B-33385 were obtained from USDA-ARS culture collection of
produce production lines effectively to ensure microbial safety National Center (NRRL) for Agricultural Utilization Research
of fresh produce. (Peoria, IL, United States) and were stored at −80°C in Trypticase
Stainless steel (SS) and plastics are preferably used in the Soy Broth with 0.6% Yeast Extract (TSBYE, Fisher Scientific,
fresh produce industry due to their anti-fouling ability (FDA, Fair Lawn, NJ, United  States) and 20% (v/v) glycerol. Each
2008). SS, a corrosion-resistant metal, is an excellent material frozen culture was activated in TSBYE at 35 ± 2°C for 24 ± 2 h
for food processing/packing equipment and extensively used statically, then sub-cultured in TSBYE for additional 24  ±  2  h
in food industries such as fresh apple packing facilities (Jellesen at 35 ± 2°C. The six-strain L. monocytogenes cocktail was prepared
et  al., 2006). A conveyor belt, one of the most prevalent food- by mixing equal volumes of each activated strain, then centrifuged
contact surfaces, directly contacts fresh produce and transports at 8,000  ×  g for 5  min at room temperature (22°C, RT). The
it to further processing or packing during post-harvest handling. resulting pellet was re-suspended in Modified Welshimer’s Broth
Polyvinyl chloride (PVC), low-density polyethylene (LDPE), (MWB, HiMedia, West Chester, PA, United  States) to have a
and rubber are FDA-approved food-contact substances that final population level of ~108  CFU/ml.
are extensively used as important components of conveyor belts
(FDA, 2017). The conveyor belts around the optical sorting Surface Selection, Preparation, and
lines have been determined to be  the major contamination Conditioning
sites in a minimally processed vegetable plant (Meireles et  al., The SS (AISI 316, No. 4 brushed finish) was obtained from
2017). The brush bed, mostly made of polyester (PET), is an the Washington State University Engineering Shops (Pullman,
important and essential processing tool of the packing lines WA, United States). PVC, LDPE, and PET sheets were purchased
of fresh apples and other fruits. The contaminated brush-bed from Interstate Plastics (Sacramento, CA, United  States), and
spray bar system was implicated in a recent caramel apple L. silicone rubber sheet was purchased from Rubber Sheet
monocytogenes outbreak (Angelo et al., 2017). L. monocytogenes Warehouse (Los Angeles, CA, United States). All surface materials
form biofilms on SS, PVC, LDPE, PET, and rubber surfaces were cut into coupons of 15  mm  ×  7.5  mm at the Washington
(Krysinski et  al., 1992; Beresford et  al., 2001; Takahashi et  al., State University Engineering Shops.
2010; Doijad et  al., 2015; Papaioannou et  al., 2018), exerting To clean coupons, the prepared surface coupons were
enhanced resistances to acid and sanitizer treatments (Ibusquiza immersed in 100% methanol (Fisher Scientific) for 1  h, rinsed
et  al., 2011; van der Veen and Abee, 2011), which makes with sterile water three times, then immersed for 1  h in 70%
routine disinfection in a food processing facility more difficult. ethanol (Fisher Scientific). The treated coupons were air dried
Food-contact surfaces are cleaned and disinfected daily with under a biosafety cabinet overnight, which were ready for
different chemical sanitizers in fresh produce processing plants biofilm growth. To condition surface coupon with organic
and apple packing facilities. Peroxyacetic acid (PAA) is an matter, the above cleaned surface coupons were immersed in
environment-friendly sanitizer that decomposes and produces 1:10 diluted apple juice or milk for 1  h at RT (Brown et  al.,
no harmful by-product (Dell'Erba et  al., 2007). Quaternary 2014). After removing conditioning solution, coupons were air
ammonium compound (QAC) and chlorine are the most dried for 1  h at RT under a biosafety cabinet.
commonly used sanitizers for surface disinfections (Robbins
et  al., 2005; Olszewska et  al., 2016; Dhowlaghar et  al., 2018).
Chlorine dioxide is considered as an alternative for chlorine L. monocytogenes Biofilm Formation
due to its high oxidizing capacity (~2.5 times higher than The above prepared coupons were subjected to a 15-min UV
that of chlorine) (Benarde et  al., 1965). The bactericidal effects treatment in the biosafety hood to surface decontamination before
of the aforementioned sanitizers against L. monocytogenes inoculation with 2.0  ml of L. monocytogenes cocktail suspension
biofilms on polystyrene surfaces were compromised in the in MWB (~108 CFU/ml). The inoculated coupons in 24-well plates
presence of organic matter or when biofilm was aged (Korany were incubated statically at RT for 7 days to grow L. monocytogenes
et  al., 2018). Different food-contact surfaces have unique biofilms without agitation (Abeysundara et  al., 2018).
physicochemical properties and hydrophobicity, which may
provide unique harbor sites for L. monocytogenes during sanitizer Sanitizer Intervention Against
intervention. Therefore, the objective of this study was to L. monocytogenes Biofilms
evaluate antimicrobial efficacies of four FDA-approved sanitizers Bioside HS (EnviroTech, Modesto, CA, United States) containing
against aged L. monocytogenes biofilms on major food-contact 15% PAA was used to prepare 160 and 200 ppm PAA solutions
surfaces in the absence or presence of organic matter. using sterile water. STOPIT (Pace International, Wapato, WA,
United  States) was diluted with sterile water to prepare 200
and 400  ppm QAC solutions. Chlorine solutions at 100 and
MATERIALS AND METHODS 200 ppm were made from Accu-Tab (Pace International, Wapato,
WA, United  States), while 2.5 and 5.0  ppm chlorine dioxide
L. monocytogenes Strains and Cocktail solutions were generated on-site using chlorine dioxide generator
Preparation donated by Pace International (Wapato, WA, United  States).
Listeria monocytogenes strain NRRL B-33069, NRRL B-57618, Concentration of PAA was verified using a AquaPhoenix
NRRL B-33006, NRRL B-33466, NRRL B-33071, and NRRL Preacetic Acid test kit (Hanover, PA, United  States), levels of

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Hua et al. Sanitizer Disinfection of L. monocytogenes

QAC and chlorine were confirmed by the QAC and Chlorine Each experiment was repeated three times independently. Data
test kits from LaMotte (Chestertown, MD, United  States), and were presented as an average from three independent studies
the concentration of chlorine dioxide solutions were measured and mean  ±  standard error mean (SEM) was reported.
by a HACH Chlorine Dioxide test kit (Loveland, CO,
United  States).
To evaluate the antimicrobial efficacy of sanitizers, 7-day-old RESULTS
L. monocytogenes biofilms on each surface coupon were washed
with 2.0  ml of sterile phosphate buffered saline (PBS) three Efficacy of Quaternary Ammonium
times and then immersed in 2.0  ml of each sanitizer solution Compound Against L. monocytogenes
for 1 or 5  min at RT. Coupons were first rinsed with 2.0  ml Biofilms on Food-Contact Surfaces
of Dey-Engley Neutralizing Broth (Oxoid, United  States), then In general, increasing the QAC concentration from 200 to
2.0  ml sterile PBS immediately after sanitizer treatment. Four 400  ppm improved its efficacy against L. monocytogenes
replicates were used for each surface material and sanitizer biofilms on different food-contact surfaces except LDPE
treatment, and triple independent experiments were conducted surface for both 1- and 5-min exposures (Figure 1). A
for each treatment combination. 5-min exposure of QAC at 200 or 400 ppm showed a similar
efficacy against L. monocytogenes biofilms on SS coupons
(Figure 1A). Except for rubber surface, the efficacy of QAC
Biofilm Detachment and Enumeration against L. monocytogenes biofilms on different surfaces was
To detach and enumerate the L. monocytogenes cells in
enhanced when exposure time increased from 1 to 5  min
biofilm on the above treated coupons, the coupon in the
(Figure 1). Among all surfaces, QAC at 5  min exposure
respective well was transferred to 2-ml microtube containing
was the most effective against L. monocytogenes biofilms
1.0  ml of sterile PBS and 3~4 glass beads. The tubes
on SS (Figure 1A), least effective against L. monocytogenes
containing coupons were vigorously vortexed for 2  min
biofilms on rubber (Figure 1E), while exhibiting a comparable
using a benchtop mixer at the maximal speed. The detached
efficacy against L. monocytogenes biofilms on LDPE and
bacterial suspension was 10-fold serially diluted with sterile
PET (Figures 1B–D). For L. monocytogenes biofilms on PVC
PBS, and appropriate dilution was plated on TSAYE plates
surface, the 5-min exposure of 400  ppm QAC showed a
in duplicate. The plates were incubated at 35  ±  2°C for
similar efficacy as those of LDPE and PET; however, 200 ppm
48  h before enumeration.
QAC for 5 min of exposure was less effective on PVC surface
than those of LDPE and PET (Figures 1B–D). QAC at the
Statistical Analysis FDA-approved concentration of 400  ppm for 5  min caused
Data were analyzed by uncorrected Fisher’s Least Significant 3.7, 3.2, 3.7, 3.6, and 3.0 log10 CFU/coupon reductions of
Difference (LSD) to determine significant difference among groups L. monocytogenes biofilms on SS, LDPE, PVC, PET, and
at p ≤ 0.05 using Prism (Version 7.0, San Diego, CA, United States). rubber surface, respectively (Figure 1).

A B

C D E

FIGURE 1  |  Antimicrobial efficacy of quaternary ammonium compound (QAC) against L. monocytogenes biofilms on food-contact surfaces. (A) Stainless steel
(SS); (B) low-density polyethylene (LDPE); (C) polyvinyl chloride (PVC); (D) polyester (PET); (E) rubber. The 7-day-old biofilms on different surface coupons
(15 mm × 7.5 mm) were treated with 200 or 400 ppm QAC for 1 or 5 min at 22°C. The surviving bacteria were shown as means ± SEMs, n = 3. a–dBars topped
with the different letters are significantly different at p ≤ 0.05.

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Hua et al. Sanitizer Disinfection of L. monocytogenes

Efficacies of Chlorine and Chlorine Dioxide (Figure 4). One min treatment of 160  ppm PAA reduced
Against L. monocytogenes Biofilms on ~4.3, 3.5, 3.8, 4.1, and 3.7 log10 CFU/coupon L. monocytogenes
Food-Contact Surfaces biofilms on SS, LDPE, PVC, PET, and rubber surfaces, respectively
Chlorine dioxide solution at 2.5 ppm exhibited a limited efficacy (Figure 4). In general, the bactericidal effects of PAA against
against L. monocytogenes biofilms on all surfaces tested; 1-min L. monocytogenes biofilms on all surfaces was not improved when
treatments only reduced ~1.1, 0.6, 0.9, 1.1, and 0.9 log10 CFU/ the PAA concentration increased from 160 to 200  ppm or when
coupon L. monocytogenes biofilms on SS, LDPE, PVC, PET, the treatment time increased from 1 to 5  min (Figure 4). The
and rubber surfaces, respectively (Figure 2). Though the efficacy 5-min treatment of 200  ppm PAA caused 4.5, 4.0, 4.4, 4.3, and
of chlorine dioxide was enhanced with increased concentration 4.4 log10 CFU/coupon reductions of L. monocytogenes biofilms
and contact time, it displayed limited potency to inactivate L. on SS, PET, PVC, LDPE, and rubber, respectively (Figure 4).
monocytogenes biofilms on food-contact surfaces. A 5-min
treatment of 5.0 ppm chlorine dioxide caused similar bactericidal Effects of Organic Matter on
efficacy against L. monocytogenes biofilms on all surfaces with Sanitizer’s Efficacy
2.4–2.7 log10 CFU/coupon reductions (Figure 2). The anti-Listeria efficacies of all sanitizers were compromised
The efficacy of chlorine against L. monocytogenes biofilms by organic matter regardless of surfaces tested; food residues
on the tested surfaces was enhanced at increased concentration from apple juice or milk comparably impacted QAC efficacy
and extended contact time except LDPE surface (Figure 3). (Figure 5). Soiling has a greater influence on the antimicrobial
A 1-min treatment of 100  ppm chlorine showed a similar efficacy of QAC against biofilms on SS and rubber than those
efficacy against L. monocytogenes biofilms as 1-min exposure on LDPE, PET, and PVC (Figure 5A). Among all tested surfaces,
of 200  ppm QAC (Figure 1) and was more effective than the anti-Listeria efficacy of chlorine on SS is the most impacted
1-min treatment of 2.5 ppm chlorine dioxide (Figure 2), causing by organic matter. Chlorine at 200  ppm and 5-min contact time
1.0–2.0 log10 CFU/coupon reductions of biofilms on all surfaces showed a similar anti-Listeria efficacy on soiled SS, LDPE and
tested. Chlorine at 200  ppm for 5.0-min exposure caused 3.8, rubber surfaces regardless of organic matter type (Figure 5B).
2.7, 3.3, 3.6, and 3.0 log10 CFU/coupon reductions of L. The bactericidal effect of chlorine dioxide against L. monocytogenes
monocytogenes biofilms on SS, LDPE, PVC, PET, and rubber biofilms was compromised by organic matter regardless of surface
surfaces, respectively (Figure 3). materials or food residue source. Chlorine dioxide at 5.0  ppm
for 5 min caused 1.0–2.0 log10 CFU/coupon reduction depending
Efficacy of Peroxyacetic Acid Against on surface material (Figure 5C). Though the PAA efficacy against
L. monocytogenes Biofilms on L. monocytogenes biofilms on all surfaces was impaired by organic
Food-Contact Surfaces soiling as much as other sanitizers, it was still the most effective
Among all selected sanitizers, PAA was the most effective sanitizer, which caused 3.0–3.7 log10 CFU/coupon reductions of
against L. monocytogenes biofilms on all food-contact surfaces L. monocytogenes biofilms on different surfaces (Figure 5D).

A B

C D E

FIGURE 2  |  Antimicrobial efficacy of chlorine dioxide against L. monocytogenes biofilms on food-contact surfaces. (A) Stainless steel (SS); (B) low-density
polyethylene (LDPE); (C) polyvinyl chloride (PVC); (D) polyester (PET); (E) rubber. The 7-day-old biofilms on different surface coupons (15 mm × 7.5 mm) were
treated with 2.5 or 5.0 ppm chlorine dioxide solution for 1 or 5 min at 22°C. The remaining bacteria post-sanitizer treatment were shown as means ± SEMs, n = 3.
a–d
Bars topped with the different letters are significantly different at p ≤ 0.05.

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Hua et al. Sanitizer Disinfection of L. monocytogenes

A B

C D E

FIGURE 3  |  Antimicrobial efficacy of chlorine against L. monocytogenes biofilms on food-contact surfaces. (A) Stainless steel (SS); (B) low-density polyethylene
(LDPE); (C) polyvinyl chloride (PVC); (D) polyester (PET); (E) rubber. The 7-day-old biofilms on different surface coupons (15 mm × 7.5 mm) were treated with 100 or
200 ppm chlorine solution for 1 or 5 min at 22°C. The survivors post-chlorine treatment were enumerated and shown as means ± SEMs, n = 3. a–cBars topped with
the different letters are significantly different at p ≤ 0.05.

A B

C D E

FIGURE 4  |  Antimicrobial efficacy of peroxyacetic acid (PAA) against L. monocytogenes biofilms on food-contact surfaces. (A) Stainless steel (SS); (B) low-density
polyethylene (LDPE); (C) polyvinyl chloride (PVC); (D) polyester (PET); (E) rubber. The 7-day-old biofilms on different surface coupons (15 mm × 7.5 mm) were
treated with 160 or 200 ppm PAA for 1 or 5 min at 22°C. The surviving bacteria were shown as means ± SEMs, n = 3. a,bBars topped with the different letters are
significantly different at p ≤ 0.05.

DISCUSSION interventions showed limited efficacies against aged L.

monocytogenes biofilms on different food-contact surfaces, but
The Effect of Concentration, Contacting their efficacies were enhanced with increased concentrations,
Time of Sanitizers on Inactivation of which was consistent with our previous findings on polystyrene
L. monocytogenes surface (Korany et  al., 2018) and other studies on SS surface
The concentrations of QAC, chlorine dioxide, chlorine and PAA (Robbins et  al., 2005; Trinetta et  al., 2012; Dhowlaghar et  al.,
against L. monocytogenes biofilms on common food-contact surfaces 2018). The antimicrobial efficacies of QAC, chlorine dioxide, and
were selected complying with FDA regulation (FDA, 2017). The chlorine at selected concentrations were improved when increasing
200  ppm QAC, 2.5  ppm chlorine dioxide, or 100  ppm chlorine contact time from 1 to 5  min, which is supported by a recent

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Hua et al. Sanitizer Disinfection of L. monocytogenes

A B

C D

FIGURE 5  |  Efficacy of four commonly used sanitizers against L. monocytogenes biofilms on food-contact surfaces conditioned with organic matters. (A)
Quaternary ammonium compound (QAC, 400 ppm); (B) chlorine (200 ppm); (C) chlorine dioxide (ClO2, 5.0 ppm); (D) peroxyacetic acid (PAA, 200 ppm); stainless
steel (SS); low-density polyethylene (LDPE); polyester (PET); polyvinyl chloride (PVC). Apple juice: food-contact surfaces were conditioned with apple juice; milk:
food-contact surfaces were conditioned with milk. The 7-day-old biofilms on different surface coupons (15 mm × 7.5 mm) were treated with the respective sanitizers
for 5 min, then survivors were enumerated and shown as means ± SEMs, n = 3. a–cBars topped with the different letters are not significantly different at p ≤ 0.05.

report of QAC and chlorine against L. monocytogenes biofilms on rubber surface was more difficult to remove by chlorine,
on SS surface (Dhowlaghar et  al., 2018). Similarly, the efficacy QAC, and chlorine dioxide than that on SS surface (Ronner
of chlorine dioxide in aqueous and gaseous phase against L. and Wong, 1993; Park and Kang, 2017). Different from QAC
monocytogenes biofilms on food contact surfaces increased with and chlorine, the anti-Listeria effects of PAA and chlorine
extended contact time (Vaid et  al., 2010; Trinetta et  al., 2012; dioxide were minimally influenced by surface material at different
Park and Kang, 2017). Increasing PAA concentration from 160 concentration and time combinations. Regardless of surfaces,
to 200  ppm or extending the contacting time from 1 to 5  min chlorine dioxide at 5.0  ppm showed a 2.5 log reduction after
at selected concentration did not improve its efficacy in general. 5-min treatment, which is a very limited efficacy in contrast
A similar result was obtained for L. monocytogenes biofilms on to 4.0 or more reduction caused by 200  ppm PAA at 5-min
polystyrene surfaces (Korany et  al., 2018). Compared with QAC, contact. Similar to our results, the aerosolized PAA exhibited
chlorine, and chlorine dioxide, PAA tested in the present study similar antimicrobial efficacy against L. monocytogenes biofilms
was the most effective sanitizer against aged L. monocytogenes on SS and PVC surfaces, though the efficacy was lower than
biofilms on all surfaces, which was consistent with findings on our finding (Park et  al., 2012). Each type of surface material
polystyrene (Korany et  al., 2018), SS (Dhowlaghar et  al., 2018), has different topography and roughness that provide unique
and PVC (Berrang et  al., 2008). It could be  due to its high microcracks/harbor sites for L. monocytogenes and protect the
reactivity, oxidizing capacity, decomposition rate, and low molecular entrapped cells from antimicrobial agents (Chaturongkasumrit
weight, which together allow PAA to penetrate biofilm matrix, et al., 2011; Schlisselberg and Yaron, 2013), which might explain
thus accomplishing bactericidal activity (Ibusquiza et  al., 2011). the difference in efficacy against biofilms on different surfaces.
In support, 20 ppm gaseous chlorine dioxide was more effective
Effects of Surface Materials on Efficacy against attached L. monocytogenes on glossy SS than coarse
SS, and Salmonella biofilms on smooth SS were more susceptible
of Different Sanitizers Against
to 50  ppm chlorine treatment than those on a rough surface
L. monocytogenes (Schlisselberg and Yaron, 2013). Surface materials with different
The efficacies of sanitizers against aged L. monocytogenes biofilms
hydrophobicity and hydration levels lead to various sanitizing
varied on different surfaces. The 1-min treatment of QAC or
efficacy; hydrophobic surface was more difficult to clean than
chlorine at selected concentrations caused comparative efficacies
hydrophilic surface (Park and Kang, 2017).
against L. monocytogenes biofilms on SS, PET, and rubber,
which is supported by a previous report on polystyrene surface
(Korany et  al., 2018). Compared with rubber and LDPE, The Antimicrobial Efficacy of Sanitizers in
400  ppm QAC and 200  ppm chlorine at 5-min exposure were the Presence of Organic Matter
more effective against L. monocytogenes biofilms on SS and Food residues established on food-contact surfaces alter the
other surfaces. In support of our finding, L. monocytogenes physicochemical property of these surfaces and impact sanitizer

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Hua et al. Sanitizer Disinfection of L. monocytogenes

efficacy (Abban et  al., 2012; Brown et  al., 2014). The present to sanitizer interventions and provide useful information for
study indicated that organic soiling, regardless of sources, food industries in selecting appropriate sanitizers for food-
impaired efficacies of all sanitizers against biofilms on all food- contact surfaces’ decontamination.
contact surfaces, which is consistent with the finding on
polystyrene surface (Korany et  al., 2018). In agreement with
our findings, protein and fat residues on SS reduced the efficacies DATA AVAILABILITY STATEMENT
of chlorine dioxide (Vandekinderen et  al., 2009), hydrogen
peroxide (Moretro et  al., 2019), acidic electrolyzed water and The datasets generated for this study are available on request
sodium hypochlorite (Ayebah et  al., 2006), QAC, chlorine, and to the corresponding author.
PAA (Aarnisalo et  al., 2000; Somers and Wong, 2004; Kuda
et al., 2008) against L. monocytogenes biofilms. Besides attracting
bacterial cells as an adhesive layer, protein coating reduced AUTHOR CONTRIBUTIONS
water contact angle, leading to decreased hydrophobicity of
food-contact surface (Abban et al., 2012; Park and Kang, 2017). ZH and AK conducted the experiments. ZH wrote the manuscript.
In addition, sanitizers may have difficulty reaching bacterial M-JZ designed the study. M-JZ and SE-S revised the manuscript.
cells due to the physical and chemical barriers built up by
exopolysaccharide substance of biofilm matrix together with
food residues (Fernandes et  al., 2015). FUNDING
This study was supported by Washington Tree Fruit
CONCLUSION Research Commission.

The type of surface material has more dramatic effects on

anti-Listeria efficacy of QAC and chlorine than those treated ACKNOWLEDGMENTS
with chlorine dioxide and PAA. Food residue soiling, regardless
of sources, reduced anti-Listeria efficacies of all sanitizers against We acknowledge Pace International Inc. for their generous
biofilms on surfaces in general. Among all sanitizers, PAA donations of Accu-Tab, Bioside HS, STOP-IT, and the Chlorine
was the most effective sanitizer against L. monocytogenes biofilms Dioxide Generator. We  would like to express our gratitude
on different surfaces. A 5-min treatment of 200  ppm PAA to Dr. Ines Hanrahan at Washington Tree Fruit Research
resulted in 3.0–3.7 log10 reductions of aged multi-strain L. for her input from a practical industry perspective. We  thank
monocytogenes biofilms on major food contact surfaces in the Mrs. Tonia Green for her assistance in preparation of experimental
presence of organic matter. Data once again highlight the materials and Mr. Mike Taylor for his critical reading of
importance of thorough cleaning of food-contact surfaces prior the manuscript.

Berrang, M. E., Frank, J. F., and Meinersmann, R. J. (2008). Effect of chemical

REFERENCES sanitizers with and without ultrasonication on Listeria monocytogenes as a
biofilm within polyvinyl chloride drain pipes. J. Food Prot. 71, 66–69. doi:
Aarnisalo, K., Salo, S., Miettinen, H., Suihko, M. L., Wirtanen, G., Autio, T., 10.4315/0362-028X-71.1.66
et al. (2000). Bactericidal efficiencies of commercial disinfectants against Brown, H. L., Reuter, M., Salt, L. J., Cross, K. L., Betts, R. P., and van Vliet,
Listeria monocytogenes on surfaces. J. Food Saf. 20, 237–250. doi: 10.1111/ A. H. M. (2014). Chicken juice enhances surface attachment and biofilm
j.1745-4565.2000.tb00302.x formation of Campylobacter jejuni. Appl. Environ. Microbiol. 80, 7053–7060.
Abban, S., Jakobsen, M., and Jespersen, L. (2012). Attachment behaviour of doi: 10.1128/AEM.02614-14
Escherichia coli K12 and Salmonella typhimurium P6 on food contact surfaces CDC (2012). Multistate outbreak of listeriosis linked to whole cantaloupes from
for food transportation. Food Microbiol. 31, 139–147. doi: 10.1016/j.fm.2012.04.003 Jensen farms, Colorado (final update). Available at: https://www.cdc.gov/
Abeysundara, P. D. A., Dhowlaghar, N., Nannapaneni, R., Schilling, M. W., listeria/outbreaks/cantaloupes-jensen-farms/index.html (Accessed July 20, 2019).
Mahmoud, B., Sharma, C. S., et al. (2018). Salmonella enterica growth and CDC (2015a). Multistate outbreak of listeriosis linked to commercially produced,
biofilm formation in flesh and peel cantaloupe extracts on four food-contact prepackaged caramel apples made from Bidart bros, apples (final update).
surfaces. Int. J. Food Microbiol. 280, 17–26. doi: 10.1016/j.ijfoodmicro.2018.04.042 Available at: https://www.cdc.gov/listeria/outbreaks/caramel-apples-12-14/index.
Angelo, K. M., Conrad, A. R., Saupe, A., Dragoo, H., West, N., Sorenson, A., et al. html (Accessed July 20, 2019).
(2017). Multistate outbreak of Listeria monocytogenes infections linked to whole CDC (2015b). Wholesome soy products, Inc. sprouts and investigation of human
apples used in commercially produced, prepackaged caramel apples: United States, listeriosis cases (final update). Available at: https://www.cdc.gov/listeria/
2014-2015. Epidemiol. Infect. 145, 848–856. doi: 10.1017/S0950268816003083 outbreaks/bean-sprouts-11-14/index.html (Accessed July 20, 2019).
Ayebah, B., Hung, Y. C., Kim, C., and Frank, J. F. (2006). Efficacy of electrolyzed CDC (2016a). Multistate outbreak of listeriosis linked to frozen vegetables
water in the inactivation of planktonic and biofilm Listeria monocytogenes (final update). Available at: https://www.cdc.gov/listeria/outbreaks/frozen-
in the presence of organic matter. J. Food Prot. 69, 2143–2150. doi: 10.4315/ vegetables-05-16/index.html (Accessed July 20, 2019).
0362-028X-69.9.2143 CDC (2016b). Multistate outbreak of listeriosis linked to packaged salads
Benarde, M. A., Israel, B. M., Olivieri, V. P., and Granstrom, M. L. (1965). Efficiency produced at Springfield, Ohio dole processing facility (final update). Available
of chlorine dioxide as a bactericide. Appl. Environ. Microbiol. 13, 776–780. at: https://www.cdc.gov/listeria/outbreaks/bagged-salads-01-16/index.html
Beresford, M. R., Andrew, P. W., and Shama, G. (2001). Listeria monocytogenes (Accessed July 20, 2019).
adheres to many materials found in food-processing environments. J. Appl. Chaturongkasumrit, Y., Takahashi, H., Keeratipibul, S., Kuda, T., and Kimura, B.
Microbiol. 90, 1000–1005. doi: 10.1046/j.1365-2672.2001.01330.x (2011). The effect of polyesterurethane belt surface roughness on Listeria

Frontiers in Microbiology | www.frontiersin.org 7 November 2019 | Volume 10 | Article 2462

Hua et al. Sanitizer Disinfection of L. monocytogenes

monocytogenes biofilm formation and its cleaning efficiency. Food Control 22, mono-species and mixed-culture simulated fish processing conditions and
1893–1899. doi: 10.1016/j.foodcont.2011.04.032 chemical disinfection challenges. Int. J. Food Microbiol. 267, 9–19. doi:
Dell’Erba, A., Falsanisi, D., Liberti, L., Notarnicola, M., and Santoroa, D. (2007). 10.1016/j.ijfoodmicro.2017.12.020
Disinfection by-products formation during wastewater disinfection with Park, S. H., Cheon, H. L., Park, K. H., Chung, M. S., Choi, S. H., Ryu, S.,
peracetic acid. Desalination 215, 177–186. doi: 10.1016/j.desal.2006.08.021 et al. (2012). Inactivation of biofilm cells of foodborne pathogen by
Dhowlaghar, N., Abeysundara, P. D., Nannapaneni, R., Schilling, M. W., aerosolized sanitizers. Int. J. Food Microbiol. 154, 130–134. doi: 10.1016/j.
Chang, S., Cheng, W. H., et al. (2018). Growth and biofilm formation by ijfoodmicro.2011.12.018
Listeria monocytogenes in catfish mucus extract on four food contact surfaces Park, S. H., and Kang, D. H. (2017). Influence of surface properties of produce
at 22 and 10°C and their reduction by commercial disinfectants. J. Food and food contact surfaces on the efficacy of chlorine dioxide gas for the
Prot. 81, 59–67. doi: 10.4315/0362-028X.JFP-17-103 inactivation of foodborne pathogens. Food Control 81, 88–95. doi: 10.1016/j.
Doijad, S. P., Barbuddhe, S. B., Garg, S., Poharkar, K. V., Kalorey, D. R., foodcont.2017.05.015
Kurkure, N. V., et al. (2015). Biofilm-forming abilities of Listeria monocytogenes Robbins, J. B., Fisher, C. W., Moltz, A. G., and Martin, S. E. (2005). Elimination
serotypes isolated from different sources. PLoS One 10:e0137046. doi: 10.1371/ of Listeria monocytogenes biofilms by ozone, chlorine, and hydrogen peroxide.
journal.pone.0137046 J. Food Prot. 68, 494–498. doi: 10.4315/0362-028X-68.3.494
FDA (2008). Guidance for industry: guide to minimize microbial food safety Ronner, A. B., and Wong, A. C. L. (1993). Biofilm development and sanitizer
hazards of fresh-cut fruits and vegetables. Available at: https://www.fda.gov/ inactivation of Listeria monocytogenes and Salmonella typhimurium on
food/guidanceregulation/ucm064458.htm (Accessed July 20, 2019). stainless steel and Buna-N rubber. J. Food Prot. 56, 750–758. doi: 10.4315/
FDA (2017). CFR-code of federal regulations title 21. Available at: https://www. 0362-028X-56.9.750
accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm (Accessed July 20, Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A.,
2019). Roy, S. L., et al. (2011). Foodborne illness acquired in the United  States-
Fernandes, M. D., Kabuki, D. Y., and Kuaye, A. Y. (2015). Behavior of Listeria major pathogens. Emerg. Infect. Dis. 17, 7–15. doi: 10.3201/eid1701.P11101
monocytogenes in a multi-species biofilm with Enterococcus faecalis and Schlisselberg, D. B., and Yaron, S. (2013). The effects of stainless steel finish
Enterococcus faecium and control through sanitation procedures. Int. J. Food on Salmonella typhimurium attachment, biofilm formation and sensitivity
Microbiol. 200, 5–12. doi: 10.1016/j.ijfoodmicro.2015.01.003 to chlorine. Food Microbiol. 35, 65–72. doi: 10.1016/j.fm.2013.02.005
Ibusquiza, P. S., Herrera, J. J. R., and Cabo, M. L. (2011). Resistance to Somers, E. B., and Wong, A. C. L. (2004). Efficacy of two cleaning and sanitizing
benzalkonium chloride, peracetic acid and nisin during formation of mature combinations on Listeria monocytogenes biofilms formed at low temperature
biofilms by Listeria monocytogenes. Food Microbiol. 28, 418–425. doi: 10.1016/j. on a variety of materials in the presence of ready-to-eat meat residue.
fm.2010.09.014 J. Food Prot. 67, 2218–2229. doi: 10.4315/0362-028X-67.10.2218
Jellesen, M. S., Rasmussen, A. A., and Hilbert, L. R. (2006). A review of metal Takahashi, H., Suda, T., Tanaka, Y., and Kimura, B. (2010). Cellular hydrophobicity
release in the food industry. Mater. Corros. 57, 387–393. doi: 10.1002/ of Listeria monocytogenes involves initial attachment and biofilm formation
maco.200503953 on the surface of polyvinyl chloride. Lett. Appl. Microbiol. 50, 618–625.
Korany, A. M., Hua, Z., Green, T., Hanrahan, I., El-Shinawy, S. H., El-Kholy, doi: 10.1111/j.1472-765X.2010.02842.x
A., et al. (2018). Efficacy of ozonated water, chlorine, chlorine dioxide, Trinetta, V., Vaid, R., Xu, Q., Linton, R., and Morgan, M. (2012). Inactivation
quaternary ammonium compounds and peroxyacetic acid against Listeria of Listeria monocytogenes on ready-to-eat food processing equipment by chlorine
monocytogenes biofilm on polystyrene surfaces. Front. Microbiol. 9:2296. doi: dioxide gas. Food Control 26, 357–362. doi: 10.1016/j.foodcont.2012.02.008
10.3389/fmicb.2018.02296 Vaid, R., Linton, R. H., and Morgan, M. T. (2010). Comparison of inactivation
Krysinski, E., Brown, L., and Marchisello, T. (1992). Effect of cleaners and of Listeria monocytogenes within a biofilm matrix using chlorine dioxide gas,
sanitizers on Listeria monocytogenes attached to product contact surfaces. aqueous chlorine dioxide and sodium hypochlorite treatments. Food Microbiol.
J. Food Prot. 55, 246–251. doi: 10.4315/0362-028X-55.4.246 27, 979–984. doi: 10.1016/j.fm.2010.05.024
Kuda, T., Yano, T., and Kuda, M. T. (2008). Resistances to benzalkonium chloride van der Veen, S., and Abee, T. (2011). Mixed species biofilms of Listeria
of bacteria dried with food elements on stainless steel surface. LWT Food monocytogenes and Lactobacillus plantarum show enhanced resistance to
Sci. Technol. 41, 988–993. doi: 10.1016/j.lwt.2007.06.016 benzalkonium chloride and peracetic acid. Int. J. Food Microbiol. 144,
McCollum, J. T., Cronquist, A. B., Silk, B. J., Jackson, K. A., O’Connor, K. A., 421–431. doi: 10.1016/j.ijfoodmicro.2010.10.029
Cosgrove, S., et al. (2013). Multistate outbreak of listeriosis associated with Vandekinderen, I., Devlieghere, F., Van Camp, J., Kerkaert, B., Cucu, T.,
cantaloupe. New Engl. J. Med. 369, 944–953. doi: 10.1056/NEJMoa1215837 Ragaert, P., et al. (2009). Effects of food composition on the inactivation
Meireles, A., Fulgencio, R., Machado, I., Mergulhao, F., Melo, L., and Simoes, M. of foodborne microorganisms by chlorine dioxide. Int. J. Food Microbiol.
(2017). Characterization of the heterotrophic bacteria from a minimally 131, 138–144. doi: 10.1016/j.ijfoodmicro.2009.02.004
processed vegetables plant. LWT Food Sci. Technol. 85, 293–300. doi: 10.1016/j.
lwt.2017.01.038 Conflict of Interest: The authors declare that the research was conducted in
Moretro, T., Fanebust, H., Fagerlund, A., and Langsrud, S. (2019). Whole room the absence of any commercial or financial relationships that could be  construed
disinfection with hydrogen peroxide mist to control Listeria monocytogenes as a potential conflict of interest.
in food industry related environments. Int. J. Food Microbiol. 292, 118–125.
doi: 10.1016/j.ijfoodmicro.2018.12.015 Copyright © 2019 Hua, Korany, El-Shinawy and Zhu. This is an open-access article
Olszewska, M. A., Zhao, T., and Doyle, M. P. (2016). Inactivation and induction distributed under the terms of the Creative Commons Attribution License (CC BY).
of sublethal injury of Listeria monocytogenes in biofilm treated with various The use, distribution or reproduction in other forums is permitted, provided the original
sanitizers. Food Control 70, 371–379. doi: 10.1016/j.foodcont.2016.06.015 author(s) and the copyright owner(s) are credited and that the original publication
Papaioannou, E., Giaouris, E. D., Berillis, P., and Boziaris, I. S. (2018). Dynamics in this journal is cited, in accordance with accepted academic practice. No use,
of biofilm formation by Listeria monocytogenes on stainless steel under distribution or reproduction is permitted which does not comply with these terms.

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