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Comparative Evaluation of Different Sanitizers Against Listeria monocytogenes Biofilms on Major Food-Contact Surfaces

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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 produce. This study evaluated efficacies of four commonly used chemical sanitizers at practical concentrations against L. monocytogenes biofilms on major food-contact surfaces including stainless steel, low-density polyethylene (LDPE), polyvinyl chloride (PVC), polyester (PET), and rubber. In general, efficacies against L. monocytogenes biofilms were enhanced by increasing concentrations of quaternary ammonium compound (QAC), chlorine, and chlorine dioxide, or extending treating time from 1 to 5 min. The 5-min treatments of 400 ppm QAC, 5.0 ppm chlorine dioxide, and 200 ppm chlorine reduced 3.0–3.7, 2.4–2.7, and 2.6–3.8 log10 CFU/coupon L. monocytogenes biofilms depending on surfaces. Peroxyacetic acid (PAA) at 160 and 200 ppm showed similar antimicrobial efficacies against biofilms either at 1- or 5-min contact. The 5-min treatment of 200 ppm PAA caused 4.0–4.5 log10 CFU/coupon reduction of L. monocytogenes biofilms on tested surfaces. Surface material had more impact on the efficacies of QAC and chlorine, less influence on those of PAA and chlorine dioxide, while organic matter soiling impaired sanitizer efficacies against L. monocytogenes biofilms independent of food-contact surfaces. Data from this study provide practical guidance for effective disinfection of food-contact surfaces in food processing/packing facilities.
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Frontiers in Microbiology | www.frontiersin.org 1 November 2019 | Volume 10 | Article 2462
ORIGINAL RESEARCH
published: 07 November 2019
doi: 10.3389/fmicb.2019.02462
Edited by:
Viduranga Y. Waisundara,
Australian College of Business and
Technology, SriLanka
Reviewed by:
Kihwan Park,
Chung-Ang University,
South Korea
Xuetong Fan,
UnitedStates Department of
Agriculture, UnitedStates
*Correspondence:
Mei-Jun Zhu
meijun.zhu@wsu.edu
These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 22 July 2019
Accepted: 14 October 2019
Published: 07 November 2019
Citation:
Hua Z, Korany AM, El-Shinawy SH
and Zhu M-J (2019) Comparative
Evaluation of Different Sanitizers
Against Listeria monocytogenes
Biolms on Major
Food-Contact Surfaces.
Front. Microbiol. 10:2462.
doi: 10.3389/fmicb.2019.02462
Comparative Evaluation of Different
Sanitizers Against Listeria
monocytogenes Biolms on Major
Food-Contact Surfaces
ZiHua1†, AhmedMahmoudKorany1,2†, Saadia HelmyEl-Shinawy2 and Mei-JunZhu1
*
1School 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 signicance
of cleaning and sanitizing food-contact surfaces to ensure microbial safety of fresh
produce. This study evaluated efcacies of four commonly used chemical sanitizers at
practical concentrations against L. monocytogenes biolms on major food-contact
surfaces including stainless steel, low-density polyethylene (LDPE), polyvinyl chloride
(PVC), polyester (PET), and rubber. In general, efcacies against L. monocytogenes biolms
were enhanced by increasing concentrations of quaternary ammonium compound (QAC),
chlorine, and chlorine dioxide, or extending treating time from 1 to 5min. The 5-min
treatments of 400ppm QAC, 5.0ppm chlorine dioxide, and 200ppm chlorine reduced
3.0–3.7, 2.4–2.7, and 2.6–3.8 log10 CFU/coupon L. monocytogenes biolms depending
on surfaces. Peroxyacetic acid (PAA) at 160 and 200ppm showed similar antimicrobial
efcacies against biolms either at 1- or 5-min contact. The 5-min treatment of 200ppm
PAA caused 4.0–4.5 log10 CFU/coupon reduction of L. monocytogenes biolms on tested
surfaces. Surface material had more impact on the efcacies of QAC and chlorine, less
inuence on those of PAA and chlorine dioxide, while organic matter soiling impaired
sanitizer efcacies against L. monocytogenes biolms independent of food-contact
surfaces. Data from this study provide practical guidance for effective disinfection of food-
contact surfaces in food processing/packing facilities.
Keywords: biolm, L. monocytogenes, sanitizers, food-contact surfaces, organic matter, peroxyacetic acid
INTRODUCTION
As a critical foodborne pathogen, Listeria monocytogenes causes approximately 1,600 cases of
infection and 260 cases of death annually in the United States (Scallan et al., 2011). It has
been implicated in multi-state outbreaks on fresh produce including cantaloupes (CDC, 2012),
prepackaged caramel apples (CDC, 2015a), bean sprouts (CDC, 2015b), frozen vegetables
(CDC, 2016a), and packaged salads (CDC, 2016b) since 2011. Contaminated food-contact
surfaces, packing lines, and environment are incriminated as the primary reasons linked to
L. monocytogenes outbreaks in fresh produce (McCollum et al., 2013; Angelo et al., 2017).
Hua et al. Sanitizer Disinfection of L. monocytogenes
Frontiers in Microbiology | www.frontiersin.org 2 November 2019 | Volume 10 | Article 2462
erefore, it is vital to sanitize food-contact surfaces along
produce production lines eectively to ensure microbial safety
of fresh produce.
Stainless steel (SS) and plastics are preferably used in the
fresh produce industry due to their anti-fouling ability (FDA,
2008). SS, a corrosion-resistant metal, is an excellent material
for food processing/packing equipment and extensively used
in food industries such as fresh apple packing facilities (Jellesen
et al., 2006). A conveyor belt, one of the most prevalent food-
contact surfaces, directly contacts fresh produce and transports
it to further processing or packing during post-harvest handling.
Polyvinyl chloride (PVC), low-density polyethylene (LDPE),
and rubber are FDA-approved food-contact substances that
are extensively used as important components of conveyor belts
(FDA, 2017). e conveyor belts around the optical sorting
lines have been determined to be the major contamination
sites in a minimally processed vegetable plant (Meireles etal.,
2017). e brush bed, mostly made of polyester (PET), is an
important and essential processing tool of the packing lines
of fresh apples and other fruits. e contaminated brush-bed
spray bar system was implicated in a recent caramel apple L.
monocytogenes outbreak (Angelo etal., 2017). L. monocytogenes
form biolms on SS, PVC, LDPE, PET, and rubber surfaces
(Krysinski et al., 1992; Beresford et al., 2001; Takahashi etal.,
2010; Doijad et al., 2015; Papaioannou et al., 2018), exerting
enhanced resistances to acid and sanitizer treatments (Ibusquiza
et al., 2011; van der Veen and Abee, 2011), which makes
routine disinfection in a food processing facility more dicult.
Food-contact surfaces are cleaned and disinfected daily with
dierent chemical sanitizers in fresh produce processing plants
and apple packing facilities. Peroxyacetic acid (PAA) is an
environment-friendly sanitizer that decomposes and produces
no harmful by-product (Dell'Erba et al., 2007). Quaternary
ammonium compound (QAC) and chlorine are the most
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
due to its high oxidizing capacity (~2.5 times higher than
that of chlorine) (Benarde etal., 1965). e bactericidal eects
of the aforementioned sanitizers against L. monocytogenes
biolms on polystyrene surfaces were compromised in the
presence of organic matter or when biolm was aged (Korany
et al., 2018). Dierent food-contact surfaces have unique
physicochemical properties and hydrophobicity, which may
provide unique harbor sites for L. monocytogenes during sanitizer
intervention. erefore, the objective of this study was to
evaluate antimicrobial ecacies of four FDA-approved sanitizers
against aged L. monocytogenes biolms on major food-contact
surfaces in the absence or presence of organic matter.
MATERIALS AND METHODS
L. monocytogenes Strains and Cocktail
Preparation
Listeria monocytogenes strain NRRL B-33069, NRRL B-57618,
NRRL B-33006, NRRL B-33466, NRRL B-33071, and NRRL
B-33385 were obtained from USDA-ARS culture collection of
National Center (NRRL) for Agricultural Utilization Research
(Peoria, IL, UnitedStates) and were stored at 80°C in Trypticase
Soy Broth with 0.6% Yeast Extract (TSBYE, Fisher Scientic,
Fair Lawn, NJ, United States) and 20% (v/v) glycerol. Each
frozen culture was activated in TSBYE at 35± 2°C for 24± 2 h
statically, then sub-cultured in TSBYE for additional 24 ± 2 h
at 35±2°C. e six-strain L. monocytogenes cocktail was prepared
by mixing equal volumes of each activated strain, then centrifuged
at 8,000 × g for 5 min at room temperature (22°C, RT). e
resulting pellet was re-suspended in Modied Welshimer’s Broth
(MWB, HiMedia, West Chester, PA, United States) to have a
nal population level of ~108 CFU/ml.
Surface Selection, Preparation, and
Conditioning
e SS (AISI 316, No. 4 brushed nish) was obtained from
the Washington State University Engineering Shops (Pullman,
WA, UnitedStates). PVC, LDPE, and PET sheets were purchased
from Interstate Plastics (Sacramento, CA, United States), and
silicone rubber sheet was purchased from Rubber Sheet
Warehouse (Los Angeles, CA, UnitedStates). All surface materials
were cut into coupons of 15mm×7.5 mm at the Washington
State University Engineering Shops.
To clean coupons, the prepared surface coupons were
immersed in 100% methanol (Fisher Scientic) for 1h, rinsed
with sterile water three times, then immersed for 1 h in 70%
ethanol (Fisher Scientic). e treated coupons were air dried
under a biosafety cabinet overnight, which were ready for
biolm growth. To condition surface coupon with organic
matter, the above cleaned surface coupons were immersed in
1:10 diluted apple juice or milk for 1 h at RT (Brown et al.,
2014). Aer removing conditioning solution, coupons were air
dried for 1 h at RT under a biosafety cabinet.
L. monocytogenes Biolm Formation
e above prepared coupons were subjected to a 15-min UV
treatment in the biosafety hood to surface decontamination before
inoculation with 2.0 ml of L. monocytogenes cocktail suspension
in MWB (~108CFU/ml). e inoculated coupons in 24-well plates
were incubated statically at RT for 7days to grow L. monocytogenes
biolms without agitation (Abeysundara et al., 2018).
Sanitizer Intervention Against
L. monocytogenes Biolms
Bioside HS (EnviroTech, Modesto, CA, UnitedStates) containing
15% PAA was used to prepare 160 and 200ppm PAA solutions
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
200ppm were made from Accu-Tab (Pace International, Wapato,
WA, United States), while 2.5 and 5.0 ppm chlorine dioxide
solutions were generated on-site using chlorine dioxide generator
donated by Pace International (Wapato, WA, United States).
Concentration of PAA was veried using a AquaPhoenix
Preacetic Acid test kit (Hanover, PA, UnitedStates), levels of
Hua et al. Sanitizer Disinfection of L. monocytogenes
Frontiers in Microbiology | www.frontiersin.org 3 November 2019 | Volume 10 | Article 2462
QAC and chlorine were conrmed by the QAC and Chlorine
test kits from LaMotte (Chestertown, MD, UnitedStates), and
the concentration of chlorine dioxide solutions were measured
by a HACH Chlorine Dioxide test kit (Loveland, CO,
United States).
To evaluate the antimicrobial ecacy of sanitizers, 7-day-old
L. monocytogenes biolms on each surface coupon were washed
with 2.0 ml of sterile phosphate buered saline (PBS) three
times and then immersed in 2.0 ml of each sanitizer solution
for 1 or 5 min at RT. Coupons were rst rinsed with 2.0 ml
of Dey-Engley Neutralizing Broth (Oxoid, UnitedStates), then
2.0 ml sterile PBS immediately aer sanitizer treatment. Four
replicates were used for each surface material and sanitizer
treatment, and triple independent experiments were conducted
for each treatment combination.
Biolm Detachment and Enumeration
To detach and enumerate the L. monocytogenes cells in
biofilm on the above treated coupons, the coupon in the
respective well was transferred to 2-ml microtube containing
1.0 ml of sterile PBS and 3~4 glass beads. The tubes
containing coupons were vigorously vortexed for 2 min
using a benchtop mixer at the maximal speed. The detached
bacterial suspension was 10-fold serially diluted with sterile
PBS, and appropriate dilution was plated on TSAYE plates
in duplicate. The plates were incubated at 35 ± 2°C for
48 h before enumeration.
Statistical Analysis
Data were analyzed by uncorrected Fishers Least Signicant
Dierence (LSD) to determine signicant dierence among groups
at p0.05 using Prism (Version 7.0, San Diego, CA, UnitedStates).
Each experiment was repeated three times independently. Data
were presented as an average from three independent studies
and mean ± standard error mean (SEM) was reported.
RESULTS
Efcacy of Quaternary Ammonium
Compound Against L. monocytogenes
Biolms on Food-Contact Surfaces
In general, increasing the QAC concentration from 200 to
400 ppm improved its efficacy against L. monocytogenes
biofilms on different food-contact surfaces except LDPE
surface for both 1- and 5-min exposures (Figure 1). A
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
against L. monocytogenes biofilms on different surfaces was
enhanced when exposure time increased from 1 to 5 min
(Figure 1). Among all surfaces, QAC at 5 min exposure
was the most effective against L. monocytogenes biofilms
on SS (Figure 1A), least eective against L. monocytogenes
biolms on rubber (Figure 1E), while exhibiting a comparable
efficacy against L. monocytogenes biofilms on LDPE and
PET (Figures 1B–D). For L. monocytogenes biofilms on PVC
surface, the 5-min exposure of 400 ppm QAC showed a
similar efficacy as those of LDPE and PET; however, 200ppm
QAC for 5min of exposure was less effective on PVC surface
than those of LDPE and PET (Figures 1B–D). QAC at the
FDA-approved concentration of 400 ppm for 5 min caused
3.7, 3.2, 3.7, 3.6, and 3.0 log10 CFU/coupon reductions of
L. monocytogenes biofilms on SS, LDPE, PVC, PET, and
rubber surface, respectively (Figure 1).
A
CD
E
B
FIGURE 1 | Antimicrobial efcacy of quaternary ammonium compound (QAC) against L. monocytogenes biolms 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 biolms 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 signicantly different at p0.05.
Hua et al. Sanitizer Disinfection of L. monocytogenes
Frontiers in Microbiology | www.frontiersin.org 4 November 2019 | Volume 10 | Article 2462
Efcacies of Chlorine and Chlorine Dioxide
Against L. monocytogenes Biolms on
Food-Contact Surfaces
Chlorine dioxide solution at 2.5ppm exhibited a limited ecacy
against L. monocytogenes biolms on all surfaces tested; 1-min
treatments only reduced ~1.1, 0.6, 0.9, 1.1, and 0.9 log10 CFU/
coupon L. monocytogenes biolms on SS, LDPE, PVC, PET,
and rubber surfaces, respectively (Figure 2). ough the ecacy
of chlorine dioxide was enhanced with increased concentration
and contact time, it displayed limited potency to inactivate L.
monocytogenes biolms on food-contact surfaces. A 5-min
treatment of 5.0ppm chlorine dioxide caused similar bactericidal
ecacy against L. monocytogenes biolms on all surfaces with
2.4–2.7 log10 CFU/coupon reductions (Figure 2).
e ecacy of chlorine against L. monocytogenes biolms
on the tested surfaces was enhanced at increased concentration
and extended contact time except LDPE surface (Figure 3).
A 1-min treatment of 100 ppm chlorine showed a similar
ecacy against L. monocytogenes biolms as 1-min exposure
of 200 ppm QAC (Figure 1) and was more eective than
1-min treatment of 2.5ppm chlorine dioxide (Figure 2), causing
1.0–2.0 log10 CFU/coupon reductions of biolms on all surfaces
tested. Chlorine at 200 ppm for 5.0-min exposure caused 3.8,
2.7, 3.3, 3.6, and 3.0 log10 CFU/coupon reductions of L.
monocytogenes biolms on SS, LDPE, PVC, PET, and rubber
surfaces, respectively (Figure 3).
Efcacy of Peroxyacetic Acid Against
L. monocytogenes Biolms on
Food-Contact Surfaces
Among all selected sanitizers, PAA was the most eective
against L. monocytogenes biolms on all food-contact surfaces
(Figure 4). One min treatment of 160 ppm PAA reduced
~4.3, 3.5, 3.8, 4.1, and 3.7 log10 CFU/coupon L. monocytogenes
biolms on SS, LDPE, PVC, PET, and rubber surfaces, respectively
(Figure 4). In general, the bactericidal eects of PAA against
L. monocytogenes biolms on all surfaces was not improved when
the PAA concentration increased from 160 to 200ppm or when
the treatment time increased from 1 to 5 min (Figure 4). e
5-min treatment of 200 ppm PAA caused 4.5, 4.0, 4.4, 4.3, and
4.4 log10 CFU/coupon reductions of L. monocytogenes biolms
on SS, PET, PVC, LDPE, and rubber, respectively (Figure 4).
Effects of Organic Matter on
Sanitizer’s Efcacy
e anti-Listeria ecacies of all sanitizers were compromised
by organic matter regardless of surfaces tested; food residues
from apple juice or milk comparably impacted QAC ecacy
(Figure 5). Soiling has a greater inuence on the antimicrobial
ecacy of QAC against biolms on SS and rubber than those
on LDPE, PET, and PVC (Figure 5A). Among all tested surfaces,
the anti-Listeria ecacy of chlorine on SS is the most impacted
by organic matter. Chlorine at 200ppm and 5-min contact time
showed a similar anti-Listeria ecacy on soiled SS, LDPE and
rubber surfaces regardless of organic matter type (Figure 5B).
e bactericidal eect of chlorine dioxide against L. monocytogenes
biolms was compromised by organic matter regardless of surface
materials or food residue source. Chlorine dioxide at 5.0 ppm
for 5min caused 1.0–2.0 log10 CFU/coupon reduction depending
on surface material (Figure 5C). ough the PAA ecacy against
L. monocytogenes biolms on all surfaces was impaired by organic
soiling as much as other sanitizers, it was still the most eective
sanitizer, which caused 3.0–3.7 log10 CFU/coupon reductions of
L. monocytogenes biolms on dierent surfaces (Figure 5D).
A
CD
E
B
FIGURE 2 | Antimicrobial efcacy of chlorine dioxide against L. monocytogenes biolms 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 biolms 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–dBars topped with the different letters are signicantly different at p0.05.
Hua et al. Sanitizer Disinfection of L. monocytogenes
Frontiers in Microbiology | www.frontiersin.org 5 November 2019 | Volume 10 | Article 2462
DISCUSSION
The Effect of Concentration, Contacting
Time of Sanitizers on Inactivation of
L. monocytogenes
e concentrations of QAC, chlorine dioxide, chlorine and PAA
against L. monocytogenes biolms on common food-contact surfaces
were selected complying with FDA regulation (FDA, 2017). e
200 ppm QAC, 2.5 ppm chlorine dioxide, or 100 ppm chlorine
interventions showed limited ecacies against aged L.
monocytogenes biolms on dierent food-contact surfaces, but
their ecacies were enhanced with increased concentrations,
which was consistent with our previous ndings on polystyrene
surface (Korany et al., 2018) and other studies on SS surface
(Robbins et al., 2005; Trinetta et al., 2012; Dhowlaghar et al.,
2018). e antimicrobial ecacies of QAC, chlorine dioxide, and
chlorine at selected concentrations were improved when increasing
contact time from 1 to 5 min, which is supported by a recent
A
CD
E
B
FIGURE 3 | Antimicrobial efcacy of chlorine against L. monocytogenes biolms 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 biolms 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 signicantly different at p0.05.
A
CD
E
B
FIGURE 4 | Antimicrobial efcacy of peroxyacetic acid (PAA) against L. monocytogenes biolms 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 biolms 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
signicantly different at p0.05.
Hua et al. Sanitizer Disinfection of L. monocytogenes
Frontiers in Microbiology | www.frontiersin.org 6 November 2019 | Volume 10 | Article 2462
report of QAC and chlorine against L. monocytogenes biolms
on SS surface (Dhowlaghar et al., 2018). Similarly, the ecacy
of chlorine dioxide in aqueous and gaseous phase against L.
monocytogenes biolms on food contact surfaces increased with
extended contact time (Vaid et al., 2010; Trinetta et al., 2012;
Park and Kang, 2017). Increasing PAA concentration from 160
to 200 ppm or extending the contacting time from 1 to 5 min
at selected concentration did not improve its ecacy in general.
A similar result was obtained for L. monocytogenes biolms on
polystyrene surfaces (Korany et al., 2018). Compared with QAC,
chlorine, and chlorine dioxide, PAA tested in the present study
was the most eective sanitizer against aged L. monocytogenes
biolms on all surfaces, which was consistent with ndings on
polystyrene (Korany et al., 2018), SS (Dhowlaghar et al., 2018),
and PVC (Berrang et al., 2008). It could be due to its high
reactivity, oxidizing capacity, decomposition rate, and low molecular
weight, which together allow PAA to penetrate biolm matrix,
thus accomplishing bactericidal activity (Ibusquiza et al., 2011).
Effects of Surface Materials on Efcacy
of Different Sanitizers Against
L. monocytogenes
e ecacies of sanitizers against aged L. monocytogenes biolms
varied on dierent surfaces. e 1-min treatment of QAC or
chlorine at selected concentrations caused comparative ecacies
against L. monocytogenes biolms on SS, PET, and rubber,
which is supported by a previous report on polystyrene surface
(Korany et al., 2018). Compared with rubber and LDPE,
400 ppm QAC and 200 ppm chlorine at 5-min exposure were
more eective against L. monocytogenes biolms on SS and
other surfaces. In support of our nding, L. monocytogenes
on rubber surface was more dicult to remove by chlorine,
QAC, and chlorine dioxide than that on SS surface (Ronner
and Wong, 1993; Park and Kang, 2017). Dierent from QAC
and chlorine, the anti-Listeria eects of PAA and chlorine
dioxide were minimally inuenced by surface material at dierent
concentration and time combinations. Regardless of surfaces,
chlorine dioxide at 5.0 ppm showed a 2.5 log reduction aer
5-min treatment, which is a very limited ecacy in contrast
to 4.0 or more reduction caused by 200 ppm PAA at 5-min
contact. Similar to our results, the aerosolized PAA exhibited
similar antimicrobial ecacy against L. monocytogenes biolms
on SS and PVC surfaces, though the ecacy was lower than
our nding (Park et al., 2012). Each type of surface material
has dierent topography and roughness that provide unique
microcracks/harbor sites for L. monocytogenes and protect the
entrapped cells from antimicrobial agents (Chaturongkasumrit
etal., 2011; Schlisselberg and Yaron, 2013), which might explain
the dierence in ecacy against biolms on dierent surfaces.
In support, 20ppm gaseous chlorine dioxide was more eective
against attached L. monocytogenes on glossy SS than coarse
SS, and Salmonella biolms on smooth SS were more susceptible
to 50 ppm chlorine treatment than those on a rough surface
(Schlisselberg and Yaron, 2013). Surface materials with dierent
hydrophobicity and hydration levels lead to various sanitizing
ecacy; hydrophobic surface was more dicult to clean than
hydrophilic surface (Park and Kang, 2017).
The Antimicrobial Efcacy of Sanitizers in
the Presence of Organic Matter
Food residues established on food-contact surfaces alter the
physicochemical property of these surfaces and impact sanitizer
AB
CD
FIGURE 5 | Efcacy of four commonly used sanitizers against L. monocytogenes biolms 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 biolms 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 signicantly different at p0.05.
Hua et al. Sanitizer Disinfection of L. monocytogenes
Frontiers in Microbiology | www.frontiersin.org 7 November 2019 | Volume 10 | Article 2462
ecacy (Abban et al., 2012; Brown et al., 2014). e present
study indicated that organic soiling, regardless of sources,
impaired ecacies of all sanitizers against biolms on all food-
contact surfaces, which is consistent with the nding on
polystyrene surface (Korany et al., 2018). In agreement with
our ndings, protein and fat residues on SS reduced the ecacies
of chlorine dioxide (Vandekinderen et al., 2009), hydrogen
peroxide (Moretro et al., 2019), acidic electrolyzed water and
sodium hypochlorite (Ayebah etal., 2006), QAC, chlorine, and
PAA (Aarnisalo et al., 2000; Somers and Wong, 2004; Kuda
etal., 2008) against L. monocytogenes biolms. Besides attracting
bacterial cells as an adhesive layer, protein coating reduced
water contact angle, leading to decreased hydrophobicity of
food-contact surface (Abban etal., 2012; Park and Kang, 2017).
In addition, sanitizers may have diculty reaching bacterial
cells due to the physical and chemical barriers built up by
exopolysaccharide substance of biolm matrix together with
food residues (Fernandes et al., 2015).
CONCLUSION
e type of surface material has more dramatic eects on
anti-Listeria ecacy of QAC and chlorine than those treated
with chlorine dioxide and PAA. Food residue soiling, regardless
of sources, reduced anti-Listeria ecacies of all sanitizers against
biolms on surfaces in general. Among all sanitizers, PAA
was the most eective sanitizer against L. monocytogenes biolms
on dierent surfaces. A 5-min treatment of 200 ppm PAA
resulted in 3.0–3.7 log10 reductions of aged multi-strain L.
monocytogenes biolms on major food contact surfaces in the
presence of organic matter. Data once again highlight the
importance of thorough cleaning of food-contact surfaces prior
to sanitizer interventions and provide useful information for
food industries in selecting appropriate sanitizers for food-
contact surfaces’ decontamination.
DATA AVAILABILITY STATEMENT
e datasets generated for this study are available on request
to the corresponding author.
AUTHOR CONTRIBUTIONS
ZH and AK conducted the experiments. ZH wrote the manuscript.
M-JZ designed the study. M-JZ and SE-S revised the manuscript.
FUNDING
is study was supported by Washington Tree Fruit
Research Commission.
ACKNOWLEDGMENTS
We acknowledge Pace International Inc. for their generous
donations of Accu-Tab, Bioside HS, STOP-IT, and the Chlorine
Dioxide Generator. We would like to express our gratitude
to Dr. Ines Hanrahan at Washington Tree Fruit Research
for her input from a practical industry perspective. Wethank
Mrs. Tonia Green for her assistance in preparation of experimental
materials and Mr. Mike Taylor for his critical reading of
the manuscript.
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Conflict of Interest: e authors declare that the research was conducted in
the absence of any commercial or nancial relationships that could beconstrued
as a potential conict of interest.
Copyright © 2019 Hua, Korany, El-Shinawy and Zhu. is is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY).
e use, distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms.
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Controlling Listeria in food is a major challenge, especially because it can persist for years in food processing plants. The best option to control this pathogen is the implementation of effective cleaning and disinfection procedures that guarantee the safety and quality of the final products. In addition, consumer trends are changing, being more aware of the importance of food safety and demanding natural foods, minimally processed and free of chemical additives. For this reason, the current consumption model is focusing on the development of preservatives of natural origin, from plants or microorganisms. In sum, this study aimed to evaluate the antimicrobial effectiveness of a citrus extract formulation rich in flavonoids against several L. monocytogenes and L. innocua strains, using in vitro test (agar diffusion test, minimum bactericidal concentration (MBC), and time-kill curves) and challenge test in food trials (carne mechada, salami, fresh salmon, lettuce, brine, and mozzarella cheese). The results presented in this work show that citrus extract, at doses of 5 and 10%, had a relevant antimicrobial activity in vitro against the target strains tested. Besides this, citrus extract applied on the surface of food had a significant antilisterial activity, mainly in carne mechada and mozzarella cheese, with reductions of up to eight logarithmic units with respect to the control. These results suggest that citrus extract can be considered a promising tool to improve the hygienic quality of ready-to-eat foods.
... The successful colonization of food processing plants by L. monocytogenes is enabled by its ability to adhere to all common surfaces occurring in food industry on which it can form biofilms (Møretrø and Langsrud, 2004). After successful adhesion, biofilms continue to grow and form sufficient protection mechanisms against disinfection like Chlorine dioxide (ClO 2 ) and mechanical cleaning, which are commonly used in the food processing industry (Blackman and Frank, 1996;Korany et al., 2018;Hua et al., 2019). Conventional methods of food decontamination for fresh food include peracitic acid, lactic acid, chlorination and treatment with diluted CLO 2 or ozone. ...
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... Contamination of FPEs is critical for contamination of ready-to-eat foods by L. monocytogenes, with the potential to lead to outbreaks of the severe and potentially life-threatening foodborne disease listeriosis [1][2][3]. L. monocytogenes has the ability to persist in FPEs via multiple adaptations, including its ability to grow at low temperatures, to form biofilms and tolerate sanitizers [4][5][6][7][8]. Even though such adaptive traits have been extensively investigated, the potential roles of bacteriophage (phage) resistance in the persistence of this pathogen in FPEs remains poorly understood. ...
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Listeria monocytogenes is a Gram-positive bacterial pathogen and the causative agent of listeriosis, a severe foodborne infection. L. monocytogenes is notorious for its ability to persist in food processing environments (FPEs) via a variety of adaptive traits. Even though traits such as cold tolerance, biofilm formation and sanitizer resistance have been extensively investigated for their roles in persistence of L. monocytogenes in FPEs, much less is known about resistance to bacteriophages. Previous studies explored phage resistance mechanisms in laboratory-created mutants but it is imperative to investigate phage resistance that is naturally exhibited in FPE-derived strains. Here, we integrated the analysis of whole genome sequence data from a panel of serotype 1/2a strains of sequence types 321 and 391 from turkey processing plants, with the determination of cell surface substituents required for phage adsorption and phage infection assays with the four wide-host-range phages A511, P100, 20422-1 and 805405-1. Using a specific set of recombinant phage protein probes, we discovered that phage-resistant strains lacked one or both of the serogroup 1/2-specific wall teichoic acid carbohydrate decorations, N-acetylglucosamine and rhamnose. Furthermore, these phage-resistant strains harbored substitutions in lmo1080, lmo1081, and lmo2550, which mediate carbohydrate decoration of the wall teichoic acids.
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Salmonella enterica is responsible for the highest number of foodborne disease outbreaks pertaining to cantaloupe industry. The objective of this study was to examine the growth and biofilm formation by outbreak strains of S. enterica ser. Poona (S. Poona), S. enterica ser. Stanley (S. Stanley) and S. enterica ser. Montevideo (S. Montevideo) on different food-contact processing surfaces in cantaloupe flesh and peel extracts at 22 °C and 10 °C. The generation time of all S. enterica strains tested was shorter in the high concentration (50 mg/ml) of cantaloupe extract and high temperature. In 50 mg/ml of cantaloupe flesh or peel extract, the populations of S. enterica were increased by 5 log CFU/ml in 24 h at 22 °C and 1 log CFU/ml in 72 h at 10 °C. In 2 mg/ml of cantaloupe flesh or peel extracts, the populations of S. enterica were increased by 3.5 log CFU/ml in 56 h at 22 °C, but there were no changes in 72 h at 10 °C. The biofilm production of S. enterica was greater at 50 mg/ml of cantaloupe extract and 22 °C, but no major differences (P ≥ 0.05) were found among the strains tested. In 50 mg/ml cantaloupe extract, S. enterica produced 5-6 log CFU/cm2 biofilm in 4-7 d at 22 °C and approximately 3.5-4 log CFU/cm2 in 7 d at 10 °C. In 2 mg/ml of cantaloupe extract, S. enterica produced 4-4.5 log CFU/cm2 biofilms in 4-7 d at 22 °C and 3 log CFU/cm2 in 7 d at 10 °C. Biofilm formation by S. Poona (01A4754) was lowest on buna-n rubber compared to stainless steel, polyethylene and polyurethane surfaces under the majority of conditions tested. Overall, these findings show that S. enterica strains can grow rapidly and form biofilms on different cantaloupe processing surfaces in the presence of low concentrations of cantaloupe flesh or peel extracts.
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The objective of this study was to determine the effect of strain and temperature on growth and biofilm formation by Listeria monocytogenes in high and low concentrations of catfish mucus extract on various food contact surfaces at 10 and 22°C. The second objective of this study was to evaluate the efficacy of disinfectants at recommended concentrations and contact times for removing L. monocytogenes biofilm cells from a stainless steel surface covered with catfish mucus extract. Growth and biofilm formation of all L. monocytogenes strains increased with higher concentrations of catfish mucus extract at both 10 and 22°C. When 15 μg/mL catfish mucus extract was added to 3 log CFU/mL L. monocytogenes, the biofilm levels of L. monocytogenes on stainless steel reached 4 to 5 log CFU per coupon at 10°C and 5 to 6 log CFU per coupon at 22°C in 7 days. With 375 μg/mL catfish mucus extract, the biofilm levels of L. monocytogenes on stainless steel reached 5 to 6 log CFU per coupon at 10°C and 6 to 7.5 log CFU per coupon at 22°C in 7 days. No differences ( P > 0.05) were observed between L. monocytogenes strains tested for biofilm formation in catfish mucus extract on the stainless steel surface. The biofilm formation by L. monocytogenes catfish isolate HCC23 was lower on Buna-N rubber than on stainless steel, polyethylene, and polyurethane surfaces in the presence of catfish mucus extract ( P < 0.05). Contact angle analysis and atomic force microscopy confirmed that Buna-N rubber was highly hydrophobic, with lower surface energy and less roughness than the other three surfaces. The complete reduction of L. monocytogenes biofilm cells was achieved on the stainless steel coupons with a mixture of disinfectants, such as quaternary ammonium compounds with hydrogen peroxide or peracetic acid with hydrogen peroxide and octanoic acid at 25 or 50% of the recommended concentration, in 1 or 3 min compared with use of the quaternary ammonium compounds, chlorine, or acid disinfectants alone, which were ineffective for removing all the L. monocytogenes biofilm cells.
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The progressive ability of a six-strains L. monocytogenes cocktail to form biofilm on stainless steel (SS), under fish-processing simulated conditions, was investigated, together with the biocide tolerance of the developed sessile communities. To do this, the pathogenic bacteria were left to form biofilms on SS coupons incubated at 15°C, for up to 240h, in periodically renewable model fish juice substrate, prepared by aquatic extraction of sea bream flesh, under both mono-species and mixed-culture conditions. In the latter case, L. monocytogenes cells were left to produce biofilms together with either a five-strains cocktail of four Pseudomonas species (fragi, savastanoi, putida and fluorescens), or whole fish indigenous microflora. The biofilm populations of L. monocytogenes, Pseudomonas spp., Enterobacteriaceae, H2S producing and aerobic plate count (APC) bacteria, both before and after disinfection, were enumerated by selective agar plating, following their removal from surfaces through bead vortexing. Scanning electron microscopy was also applied to monitor biofilm formation dynamics and anti-biofilm biocidal actions. Results revealed the clear dominance of Pseudomonas spp. bacteria in all the mixed-culture sessile communities throughout the whole incubation period, with the in parallel sole presence of L. monocytogenes cells to further increase (ca. 10-fold) their sessile growth. With respect to L. monocytogenes and under mono-species conditions, its maximum biofilm population (ca. 6logCFU/cm2) was reached at 192h of incubation, whereas when solely Pseudomonas spp. cells were also present, its biofilm formation was either slightly hindered or favored, depending on the incubation day. However, when all the fish indigenous microflora was present, biofilm formation by the pathogen was greatly hampered and never exceeded 3logCFU/cm2, while under the same conditions, APC biofilm counts had already surpassed 7logCFU/cm2 by the end of the first 96h of incubation. All here tested disinfection treatments, composed of two common food industry biocides gradually applied for 15 to 30min, were insufficient against L. monocytogenes mono-species biofilm communities, with the resistance of the latter to significantly increase from the 3rd to 7th day of incubation. However, all these treatments resulted in no detectable L. monocytogenes cells upon their application against the mixed-culture sessile communities also containing the fish indigenous microflora, something probably associated with the low attached population level of these pathogenic cells before disinfection (<102CFU/cm2) under such mixed-culture conditions. Taken together, all these results expand our knowledge on both the population dynamics and resistance of L. monocytogenes biofilm cells under conditions resembling those encountered within the seafood industry and should be considered upon designing and applying effective anti-biofilm strategies.
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The objective of this study was to evaluate the influence of surface properties of produce and food contact surfaces on the antimicrobial effect of chlorine dioxide (ClO2) gas against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes. The hydrophobicity of the selected surfaces was evaluated by water contact angle measurements. White light scanning interferometry (WLSI) was used to acquire surface roughness values of each surface. Produce and food contact surfaces inoculated with foodborne pathogens were treated with 20 ppmv ClO2 gas for 5, 10, and 15 min. As treatment time increased, different levels of inactivation of the three pathogens were observed among the samples. Contact angles of produce and food contact surfaces were highly and negatively correlated with the log reduction of all three pathogens. There were generally weaker correlations between the roughness values of sample surfaces and microbial reduction compared to those between hydrophobicity and microbial reduction. The results of this study showed that surface hydrophobicity is a more important factor relative to bacterial inactivation by ClO2 gas from the surface than is surface roughness. Also, the existence of crevices with features of similar size to the pathogen cell was more important than the Ra and Rq values in the inactivation of pathogens.
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The knowledge on the microorganisms present in an industrial process is crucial to delineate the best strategy for their effective control. The aims of the present work were to isolate, identify and characterize (in terms of production of proteases, gelatinases and siderophores, quorum-sensing inhibition and biofilm formation) the resident heterotrophic bacteria present in a minimally processed vegetables (MPV) plant where sodium hypochlorite was used for decontamination. A total of 47 isolates were obtained with 49% belonging to the Pseudomonas genera. Twenty different bacterial species were identified and the conveyor belt in the high care area was found to be a significant source of contamination. Most of the isolates were capable of producing virulence related molecules and all isolates were able to form biofilm. Pseudomonas was the genera with the highest biofilm formation ability, being the predominant microflora along the process chain. Even if no relevant foodborne pathogen was isolated, the results clearly propose that improvements in decontamination during processing are required to effectively control microbial presence in the final product.
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Whole apples have not been previously implicated in outbreaks of foodborne bacterial illness. We investigated a nationwide listeriosis outbreak associated with caramel apples. We defined an outbreak-associated case as an infection with one or both of two outbreak strains of Listeria monocytogenes highly related by whole-genome multilocus sequence typing (wgMLST) from 1 October 2014 to 1 February 2015. Single-interviewer open-ended interviews identified the source. Outbreak-associated cases were compared with non-outbreak-associated cases and traceback and environmental investigations were performed. We identified 35 outbreak-associated cases in 12 states; 34 (97%) were hospitalized and seven (20%) died. Outbreak-associated ill persons were more likely to have eaten commercially produced, prepackaged caramel apples (odds ratio 326·7, 95% confidence interval 32·2–3314). Environmental samples from the grower's packing facility and distribution-chain whole apples yielded isolates highly related to outbreak isolates by wgMLST. This outbreak highlights the importance of minimizing produce contamination with L. monocytogenes . Investigators should perform single-interviewer open-ended interviews when a food is not readily identified.
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Biofilm formation by seven strains of Listeria monocytogenes and one strain of Salmonella typhimurium on stainless steel and Buna-n rubber was examined under two nutrient conditions. The type of surface, nutrient level, and organism influenced biofilm development and production of extracellular materials. Buna-n had a strong bacteriostatic effect on L. monocytogenes , and biofilm formation on Buna-n under low nutrient conditions was reduced for four of the seven strains tested. Buna-n was less bacteriostatic toward S. typhimurium . It inhibited the growth of several other pathogens to varying degrees. An ethylene propylene diamine monomer rubber was less inhibitory than Buna-n, and Viton rubber had no effect. The effectiveness of sanitizers on biofilm bacteria was examined. Biofilms were challenged with four types of detergent and nondetergent sanitizers. Resistance to sanitizers was strongly influenced by the type of surface. Bacterial biofilm populations on stainless steel were reduced 3-5 log by all the sanitizers, but those on Buna-n were resistant to these sanitizers and were reduced less than 1-2 log. In contrast, planktonic (suspended) bacteria were reduced 7-8 log by these sanitizers. Chlorine and anionic acid sanitizers generally removed extracellular materials from biofilms better than iodine and quaternary ammonium detergent sanitizers. Scanning electron microscopy demonstrated that biofilm cells and extracellular matrices could remain on sanitized biofilm cells and extracellular matrices could remain surfaces from which no viable cells were recovered.