Food Microbiology 90 (2020) 103468

Contents lists available at ScienceDirect

Food Microbiology
journal homepage: www.elsevier.com/locate/fm
Prevalence of Listeria spp. in produce handling and processing facilities in T
the Pacific Northwest
Jorgensen Johna, Waite-Cusic Joyb, Kovacevic Jovanaa,∗
a
Food Innovation Center, 1207 NW Naito Parkway, Oregon State University, Portland, OR, 97209, USA
b
Department of Food Science and Technology, 100 Wiegand Hall, Oregon State University, Corvallis, OR, 97331, USA
A R T I C LE I N FO A B S T R A C T
Keywords: Listeria monocytogenes is a significant concern for the produce industry; however, there is limited information to
Environmental monitoring support the practical decision-making to mitigate this risk. This study investigated the prevalence of Listeria spp.
Produce and L. monocytogenes in seven produce handling and processing (PHP) facilities in the Pacific Northwest. PHP
Listeria facilities were defined as facilities that receive raw agricultural commodities and further handle, pack, wash, or
Listeria monocytogenes
process prior to distribution into the retail sector. Environmental swabs (n = 50/facility) were collected in high-
Prevalence
Packinghouse
risk areas (e.g., near raw product entry points) from seven PHP facilities over two visits. Listeria spp. were
isolated using modified ISO 11290-1 method and speciated with Microgen® Listeria-ID. Listeria spp., including L.
monocytogenes, were found in 5/7 PHP. Prevalence of Listeria spp. ranged from 2% to 26% in these five facilities.
Drains, entry areas, and portable equipment consistently tested positive for Listeria spp. during active produc-
tion. Two additional sampling rounds (n = 50/round) were conducted in the highest prevalence facility (Facility
#1). Overall, Listeria spp. were detected in 44/150 (29.3%) swabs collected from Facility #1. This study de-
monstrated the high prevalence of Listeria spp. near raw product entry points across PHP facilities.
1. Introduction buildings (FDA, 2019). Produce facilities face different challenges, as
they often receive produce directly after harvest and quickly handle it
The United States (U.S.) public health agenda includes a decrease in to facilitate refrigerated storage or distribution without an anti-
the annual number of foodborne illnesses, including listeriosis. The microbial intervention (i.e., kill step). There are minimal expectations
Food Safety Modernization Act (FSMA) was passed in 2011 with the or support for these facilities to mitigate the potential for L. mono-
intention of supporting this public health goal. Due to the high mor- cytogenes contamination, despite significant listeriosis outbreaks being
bidity and mortality rate of listeriosis, FSMA regulations, especially the linked to produce farm operations. One of the most significant lister-
Preventive Controls for Human Food (PCHF) rule, include emphasis on iosis outbreaks in U.S., leading to 33 deaths, was linked to con-
controlling Listeria monocytogenes in food processing facilities where taminated cantaloupe from a single farm operation (CDC, 2012). The
ready-to-eat products are openly exposed to the environment. In 2017, unhygienic production environment was likely the reason for the con-
the U.S. Food and Drug Administration (FDA) issued a new draft gui- tamination of the whole cantaloupes (McCollum et al., 2013). Similarly,
dance document, addressing best practices and control of L. mono- environmental sources of Listeria were identified in the 2015 multistate
cytogenes in ready-to-eat foods (FDA, 2017). listeriosis outbreak associated with caramel apples (Angelo et al., 2017;
The PCHF and guidance documents provide a strategy for mitigating CDC, 2015), as well as the 2016 outbreaks linked to frozen vegetables
environmental pathogens in food processing facilities; however, these and packaged salads (CDC, 2016a; CDC, 2016b).
do not apply to farm and mixed-type facility operations that handle Since Listeria spp. are saprophytes that are commonly present in the
fresh produce. Instead, these facilities are subject to the FSMA Produce soil of various agricultural landscapes (Chapin et al., 2014; Linke et al.,
Safety Rule (PSR). The PSR focuses on standards and strategies to 2014; Strawn et al., 2013; Vivant et al., 2013), freshly harvested pro-
promote the safe harvesting and manufacturing of produce, including duce, particularly crops that are in close proximity to the topsoil, are at
sprouts (FDA, 2019). Specifically, it provides standards for agricultural high risk for Listeria spp. contamination. As these crops are harvested
water, biological soil amendments, domesticated and wild animals, and transported into the packing or processing facility, they carry Lis-
worker training and health and hygiene, and equipment, tools and teria cells that have the potential to establish themselves in facilities,
∗
Corresponding author. Food Innovation Center, 1207 NW Naito Parkway, Oregon State University, Portland, OR, 97209, USA.
E-mail address: jovana.kovacevic@oregonstate.edu (K. Jovana).
https://doi.org/10.1016/j.fm.2020.103468
Received 17 September 2019; Received in revised form 17 December 2019; Accepted 19 February 2020
Available online 28 February 2020
0740-0020/ © 2020 Elsevier Ltd. All rights reserved.
J. John, et al. Food Microbiology 90 (2020) 103468
PSR indicates facility is covered under 21 CFR Parts 11, 16, and 112 Standards for the Growing, Harvesting, Packing, and Holding of Produce for Human Consumption Rule (Produce Safety Rule) or PC indicates

and lead to recurrent contamination of handled/processed produce.

Few studies have investigated the prevalence of Listeria spp. and L.
monocytogenes in produce, and produce handling and processing (PHP)
Yes; 16/week; Listeria spp. (if positive will speciate to L. monocytogenes),

environments (Gianfranceschi et al., 2002; Hoelzer et al., 2011; Leong

Salmonella spp., E. coli (generic, check if E. coli 0157:H7 if positive)

et al., 2017, 2014; Prazak et al., 2002; Sauders et al., 2009; Tan et al.,
facility is covered under 21 CFR Part 117 Current Good Manufacturing Practice, Hazard Analysis, and Risk-Based Preventive Controls for Human Food Rule (Preventive Controls), or both (PSR & PC).

2019). These studies demonstrated Listeria spp. prevalence in specific

produce associated environments and regularly combined results from
product testing, retail, and processing environments. However, data are
Yes; Listeria spp., a few times per year in drains only

lacking for the environmental prevalence of Listeria spp. in specific U.S.

Yes; Daily, varies on # of samples; Salmonella spp.

regions (e.g., Pacific Northwest, [PNW]) strictly related to the PHP

industry.
Prevalence data specific to the produce industry, and especially in
Yes; 10/month; Listeria and Salmonella

facilities covered by different regulations, as well as regional differ-

Environmental Monitoring Program

ences, would be helpful to inform future food safety strategies. The

overall objective of this study was to investigate the prevalence and
E. coli (generic), Listeria spp.
Yes; no additional details.

Yes; no additional details.

diversity of Listeria spp. in PHP facilities in the PNW states of Oregon

and Washington. A secondary objective was to identify potential routes
of Listeria spp. movement in PHP facilities.
2. Materials and methods
Facility #1 handles, washes, and packs most of these commodities; however, some are received packaged and simply stored prior to distribution.

2.1. Produce handling and processing facilities

No

Not disclosed

Not disclosed

A flyer about environmental monitoring research was distributed to

Sanitizersb

ClO−QAC

regional PHP facilities at workshops and through extension listservs. No

financial incentive was offered to encourage participation. Facilities
QAC

QAC
PAA

ClO

ClO

that responded to the participation request were contacted via email to

verify eligibility and provide additional information on the research
Cleaning and Sanitation

goals, expectations associated with participation, and anonymity. For

the purpose of this research, the definition of a PHP facility was one
that grew, harvested, packed, and/or processed produce in Oregon or
Washington state. Additional phone calls between researchers and PHP
Frequency

facility personnel were completed to gather relevant information about

Daily

Daily
Daily
Daily

Daily

Daily
Daily
their operation, to schedule site visits, sample collection dates, and to
gather other relevant information. Relevant information included
number and type of crops in facility, production details (i.e. handle,

ClO− = Hypochlorite; PAA = Peroxyacetic Acid; QAC = Quaternary Ammonium Compound.

Production (Seasonal or
pack, process produce, or combination), final products, sanitation
practices, and environmental monitoring history. Seven PHP facilities
agreed to participate in the study; relevant information about each fa-

year-round)

Year-round

Year-round
Year-round

Year-round
cility is summarized in Table 1.

Seasonal

Seasonal
Seasonal
2.2. Environmental sample collection

Produce handling and processing (PHP) facilities that participated in this study.
# of Employees
Each participating PHP facility was visited to identify and cate-
gorize environmental sampling sites to maintain consistency across

350–400
80–150
25–100
25–100

25–100
25–400
facilities. Final sampling site selections and number of samples per

30–50
sampling round are shown in Table 2. Oregon State University la-
boratory staff collected all environmental samples from each facility
within 2–3 h of the beginning of the production day. Exact sampling

FSMA Compliance
locations for each facility were documented on a facility map with a
brief description, along with a digital photograph. A supervisor from

PSR & PC

PSR & PC

PSR & PC
PSR & PC
each facility was present and actively observed environmental sample

PSR only

PC only
PC only
collection. A copy of all written information and metadata were pro-

Typea
vided to the facility prior to leaving the property. Each facility was
visited for sample collection at least twice (Rounds A and B) during the

Kill Step
2018–2019 processing year. Findings from these initial rounds in-

Yes

Yes
No

No
No

No
No
formed decisions for future site visits and additional swabbing rounds
(Rounds C and D). Environmental samples were collected by swabbing

# of Commodities
an approximate area of 930 cm2 (30.5 cm × 30.5 cm) with a sponge-
stick moistened with neutralizing buffer (3 M, St. Paul, MN, USA) on
non-food contact surfaces (NFCS) only. The area was swabbed five
times vertically with one side, and five times horizontally with one side.

48c

26
3
3
3

7
3
Sampling locations with insufficient surface area (i.e., equipment

Facility
legs) were swabbed as completely as possible. Swabs were returned to

Table 1
the original bag and held in a cooler at approximately 4 °C until all

#1

#2
#3
#4

#5

#6
#7

b
a

c
samples had been collected. Samples were transported to the Food
2
J. John, et al. Food Microbiology 90 (2020) 103468
Table 2 manufacturer's instructions. Isolates were further characterized by
Environmental samples from each sampling round and sample site description. Gram-stain reaction, catalase, and motility tests. Isolates confirmed as
Sample Site Sampling Roundsa Listeria spp. were suspended in 50% glycerol and stored at −80 °C. L.
monocytogenes ATCC 19115 and L. innocua ATCC 33090 served as re-
A B C D ference strains for morphology and test interpretation. All L. mono-
cytogenes strains from positive sampling sites (up to three from each
Drain 10 10 12–14 12–14
Forklift tireb 2 2 4 4 site) were serogrouped by PCR (n = 75). Furthermore, antibiograms of
Forklift traffic area (floor) 1 1 4 4 L. monocytogenes isolates recovered from Facility #1 (n = 40) were
Side of conveyor 1 2 5–7 5–7 assessed to further investigate potential relatedness of these strains.
Pallet or bin 2 2 2 2
Entry point 0 3 4 4
Portable items 0 5 5 5 2.4. Multiplex PCR serogrouping of Listeria monocytogenes isolates
Floor below initial productionc 0 1 1 1
Outside Surface 0 0 3 3
Coolers 0 0 4 4
A modified version of the multiplex polymerase chain reaction
Otherd 4 4 4 4 (PCR) previously described by Doumith et al. (2004) was used for
Total 20 30 50 50 molecular serogrouping of L. monocytogenes isolates (n = 75). The
multiplex PCR mixture (25 μl) consisted of 1 Unit of Taq DNA Poly-
a
All facilities were sampled twice (Rounds A and B). A single facility merase (NEB, New England BioLabs, Ipswich, MA, USA), 1X PCR buffer
(Facility #1) was sampled on two additional visits (Rounds C and D).
b mix (NEB), 200 μM dNTPs (NEB), 1 μM of each primer for lmo0737,
Front left tire.
ORF2819, and ORF2110, 1.5 μM of each primer for lmo1118, and
c
Samples taken at this site were on the floor below where raw product first
started production.
0.2 μM of each primer for prs. Primers were purchased from Integrated
d
Four sample sites for each round were chosen by facility personnel. DNA Technologies (IDT, Coralville, IA, USA) and DNA sequences are
Examples include hand wash sink drain pipes, walls, temporary equipment, shown in Table 3.
equipment outside, raw product trailers, additional drain and floor locations, PCR was performed using the Applied Biosystems™ SimpliAmp™
waste bins, hollow cracks and crevices on equipment, and concrete seams on thermocycler (Fisher Scientific, Waltham, MA, USA) with an initial
floors. denaturation step at 94 °C for 3 min; 35 cycles of denaturation at 94 °C
for 24 s, annealing at 53 °C for 75 s, and extension at 72 °C for 75 s; and
Safety Laboratory at Oregon State University's Food Innovation Center a final incubation at 72 °C for 7 min. PCR amplification products (8 μl)
and stored at 4 °C for up to 48 h prior to analysis. were separated on a 2% UltraPure™ agarose gel containing Gel Red
(Thermo Fisher Scientific, Waltham, MA, USA) in TBE buffer. Amplicon
separation was achieved using a voltage gradient program of 45 min at
2.3. Analysis of environmental samples for Listeria spp.
60 V, 80 V, and 100 V. PCR products were visualized using the Gel
Doc™ XR + Imager (Bio-Rad, Hercules, CA, USA). A 1-Kb DNA ladder
Demi-Fraser broth (45 ml; DFB, Neogen, Lansing, MI, USA) was
(Thermo Fisher Scientific, Waltham, MA, USA) served as the size
added to each environmental sample bag. Sample swabs were massaged
standard for each gel. Control strains for the following known L.
by hand or with a stomacher and incubated at 30 °C for 24 h. Following
monocytogenes serotypes were included in each gel: 1/2a, DE25-1; 1/
incubation, samples were mixed with 100 μl transferred to 10 ml of
2 b, OE90-1; 1/2c, FF1-1 and OF64-2; and 4 b, FF5-1 (Kovacevic et al.,
Fraser broth (FB, Neogen, Lansing, MI, USA) and incubated at 35 °C for
2013a).
24–48 h with shaking at 200 rpm. Aliquots (10 μl) of the incubated DFB
and/or FB were spread plated onto both a Harlequin Listeria agar ac-
cording to the formulation of Ottaviani and Agosti (ALOA, Neogen, 2.5. Antimicrobial resistance by disk diffusion
Lansing, MI, USA), and PALCAM agar (Neogen, Lansing, MI, USA);
incubated at 35 °C for 24–48 h. ALOA and PALCAM plates were eval- Antimicrobial resistance (AMR) profiles of L. monocytogenes isolates
uated for the presence of typical Listeria spp. colonies. For each sample, were determined using methods previously described (Kovacevic et al.,
up to 20 colonies displaying morphology typical for Listeria spp. were 2013b; Milillo, 2015). Briefly, each isolate was streaked for isolation on
selected and transferred by stabbing to trypticase soy agar (TSA) + 5% TSAYE (Neogen) and incubated at 35 °C for 24 h. A single colony was
defibrinated horse blood (HBA; Hardy Diagnostics, Santa Maria, CA, transferred to TSB (3 ml; Neogen) and incubated at 35 °C for 18 h with
USA). Hemolysis was evaluated after incubation at 35 °C for 24 h and at shaking (200 rpm). A 70 μl aliquot of the liquid culture was mixed with
least three colonies were streaked for isolation on TSA +5% sheep 7 mL of 0.75% agar tempered at 45 °C and overlaid onto previously
blood (BAP; Hardy Diagnostics, Santa Maria, CA, USA). Both hemolytic prepared Mueller-Hinton agar plates (MHA, Neogen). Antibiotic sensi-
and non-hemolytic colonies were selected for further identification. tivity disks (Table 4; Becton, Dickinson and Company (BD), Sparks,
Isolates were speciated using the Microgen® Listeria-ID system MD) were placed onto the surface of the solidified MHA plates and
(Microgen; Microgen Bioproducts, Camberly, UK) following incubated at 35 °C for 24 h. The diameter of each zone of inhibition was
Table 3
PCR target genes, primer sequences, and amplicon size for serogrouping Listeria monocytogenes (Adapted from Doumith et al., 2004).
Target Primer Sequence (5′-3′) Product size (bp) Serogroups resulting in PCR product
prs prsF: 5′-GCTGAAGAGATTGCGAAAGAAG-3′ 370 Listeria spp.
prsR: 5′-CAAAGAAACCTTGGATTTGCGG-3′
ORF2819 ORF2819F: 5′-AGCAAAATGCCAAAACTCGT-3′ 471 1/2 b, 3 b, 4 b, 4 d, 4e
ORF2819R: 5′-CATCACTAAAGCCTCCCATTG-3′
ORF2110 ORF2110F: 5′-AGTGGACAATTGATTGGTGAA-3′ 597 4 b, 4 d, 4e
ORF2110R: 5′-CATCCATCCCTTACTTTGGAC-3′
lmo0737 lmo0737 F: 5′-AGGGCTTCAAGGACTTACCC-3′ 691 1/2a, 1/2c, 3a, 3c
lmo0737 R: 5′-ACGATTTCTGCTTGCCATTC-3′
lmo1118 lmo1118 F: 5′-AGGGGTCTTAAATCCTGGAA-3′ 906 1/2c, 3c
lmo1118 R: 5′-CGGCTTGTTCGGCATACTTA-3′
3
J. John, et al. Food Microbiology 90 (2020) 103468
Table 4
Panel of 18 antibiotics used to assess antimicrobial resistance of L. monocytogenes recovered from one produce handling facility (Facility #1) using disk diffusion
assay. This table also includes antibiotic breakpoint ranges for control strains and break points for L. monocytogenes.
Antibiotic Abbreviation Antibiotic Disk Dose S. aureusa ATCC 25923 range E. colia ATCC 25922 range L. monocytogenes. breakpoints (mm)
(μg) (mm) (mm)
Sensitive (S) Intermediate (I) Resistant (R)
Amikacin AMK 30 20–26 19–26 ≤14 15–16 ≥17
Ampicillin AMP 10 27–35 15–22 19 -b 20
Cefoxitin FOX 30 23–29 23–29 14 15–17 18
Chloramphenicol CHL 30 19–26 21–27 12 13–17 18
Ciprofloxacin CIP 5 22–30 29–37 15 16–20 21
Clindamycin CLI 2 24–30 N/Ac 14 15–20 21
Erythromycin ERY 15 22–30 N/Ac 14 15–22 23
Gentamicin GEN 10 19–27 19–26 12 13–14 15
Imipenem IPM 10 N/Ac 26–32 13 14–15 16
Kanamycin KAN 30 19–26 17–25 13 14–17 18
Nalidixic acid NAL 30 N/Ac 22–28 13 14–18 19
Novobiocin NOV 30 22–31 N/Ac 17 18–21 22
Penicillin PEN 10 Ud 26–37 N/Ac 19 20–27 28
Rifampin RIF 5 26–34 8–10 16 17–19 20
Streptomycin STR 10 14–22 12–20 11 12–14 15
Co-trimoxazole SXT 1.25/23.75e 24–32 23–29 10 11–15 16
Tetracycline TET 30 24–30 18–25 14 15–18 19
Vancomycinf VAN 5 17–21 N/Ac 9 -b 10
a
Control strain ranges for each antibiotic determined from Clinical Laboratory Standards Institute (CLSI, Wayne, PA, USA).
b
No intermediate breakpoint, only sensitive or resistant, (−).
c
Breakpoints for L. monocytogenes. were based on CLSI (Wayne, PA, USA). Breakpoints for nalidixic acid and streptomycin were based on Enterobacteriaceae values;
vancomycin was based on Enterococcus values; all others were based on Staphylococcus values. Ranges or breakpoints not determined or available by CLSI guidelines,
(N/A).
d
Penicillin disk concentration in international units of penicillin (U).
e
Co-trimoxazole is composed of two antibiotics, trimethoprim (1.25 μg) and sulfamethoxazole (23.75 μg).
f
Vancomycin L. monocytogenes. breakpoints determined from Dalynn Biologicals (Calgary, AB, Canada).
measured to the nearest mm. Interpretation of antibiotic susceptibility Table 5
(sensitive, intermediate, resistant) was determined in accordance with Detection of Listeria spp. in environmental sampling sites at PHP facilities
Clinical Laboratory Standards Institute (CLSI, Wayne, PA, USA) criteria during initial rounds of testing (rounds A and B).
(Table 4). L. monocytogenes isolates displaying resistance to specific Facility Samples Positive Recovered Species Positive Sample Site Types
antibiotics were verified by additional replications of the disk diffusion for Listeria spp. (%) (n)
assay. Escherichia coli ATCC 35218 and Staphylococcus aureus ATCC
#1 13/50a (26%) L. innocua (3) Drain, Entry point, Floor,
25923 were used as control cultures for the disk diffusion assay. AMR
L. ivanovii (1) Other
patterns were compared for isolates recovered from the same swab L. monocytogenes (6)
sample. Isolates were considered to be unique strains if there was a L. welshimeri (5)
difference of at least 3 mm for a inhibition zone of a single antibiotic or #2 5/50 (10%) L. monocytogenes (5) Drain, Entry point, Forklift
a difference of at least 2 mm for three or more antibiotics. Otherwise, tire, Forklift traffic area
#3 1/50 (2%) L. monocytogenes (1) Drain
isolates from the same sample were considered to be representative of a #4 3/50 (6%) L. innocua (2) Drain, Equipment Leg
single strain (i.e., “clonal”) and one was chosen as the representative L. monocytogenes (1)
strain for reporting purposes. When the inhibition zones of “identical” #5 0/50 (0%) None detected
isolates spanned the resistance classification, the most resistant isolate #6 2/50 (4%) L. monocytogenes (2) Drain, Entry point
#7 0/50 (0%) None detected
was chosen.
Total 24/350 (6.8%) 4 species 7 types of sample sites
a
2.6. Statistical analysis Two of the samples collected from Facility #1 were positive for both L.
monocytogenes and L. welshimeri.
Significant differences in the prevalence of Listeria spp. between
facilities were determined using chi-square test of independence using spp. (13/50; 26%). Facility #1 contributed > 50% of the positive
GraphPad QuickCalcs (https://www.graphpad.com/quickcalcs/). samples for the entire study, and it was the only facility where L. wel-
shimeri and L. ivanovii were detected. Due to this high prevalence, Fa-
cility #1 was the focus of subsequent rounds of sampling (rounds C and
3. Results
D). The survey in Table 1 reports facility characteristics that may
contribute to the high Listeria spp. prevalence observed in Facility #1.
3.1. Prevalence of Listeria spp. in PNW PHP facilities
Notably, this facility washes and packs (no processing or kill-step)
nearly 50 different commodities year-round, drastically higher than
Following two rounds of sampling, Listeria spp. were isolated from
Facility #7 that processes three commodities seasonally (6–8 months/
environmental samples in 5/7 (71%) PHP facilities (Table 5). Facilities
year) and has an active environmental monitoring program for Listeria
significantly differed in the prevalence of Listeria spp. in environmental
spp. Facility #7 had no positives for Listeria spp. Though no significant
samples (p < 0.0001). Listeria spp. were not detected in any of the
correlations were observed between facility characteristics (Table 1)
environmental samples collected from Facilities #5 and #7. Facilities
and positive samples, examining general facility characteristics pro-
#2, #3, #4, and #6 had low percentages (≤10%) of environmental
vided information that may support the variations observed in Listeria
samples that were positive for Listeria spp., whereas Facility #1 had the
spp. prevalence among facilities.
highest number of environmental samples that were positive for Listeria
4
J. John, et al. Food Microbiology 90 (2020) 103468
Table 6 positive for Listeria spp. included drains (n = 5), the floor underneath
Prevalence of Listeria monocytogenes serogroups from produce handling and raw product intake (n = 1), and the floor near facility entry points
packing facilities (PHP) in the Pacific Northwest. (n = 1). The remaining positive sampling sites were associated with a
Facility ID No. of L. monocytogenes (%) raw produce trailer that was driven into the facility to expedite un-
loading and sorting of raw product. A trailer tire (n = 1) and the floor
Serogroup 1 (1/2a, 3a) Serogroup 4 (4 b, 4 d, 4e) near the trailer (n = 2) were positive for Listeria spp. Four Listeria
species were isolated from this round of sampling: L. welshimeri (n = 5),
#1 12/52 (23%) 40/52 (77%)
#2 0/9 (0%) 9/9 (100%) L. monocytogenes (n = 5), L. innocua (n = 1) and L. ivanovii (n = 1). L.
#3 0/3 (0%) 3/3 (100%) welshimeri was recovered from a single trench drain (drain #1) at three
#4 2/2 (100%) 0/3 (0%) different locations, the raw product trailer tire (n = 1), and the floor
#6 9/9 (100%) 0/9 (0%)
below the raw product trailer (n = 1). L. monocytogenes was recovered
Total 23/75 (31%) 52/75 (69%)
from two trench drains (drains #1 and #2) at single locations per drain.
It was also recovered from the floor near an entry point, the raw pro-
Across all produce facilities, 11 and 15 environmental samples were duct trailer tire, and the floor surrounding the raw product trailer. L.
positive for Listeria spp. (L. innocua, L. ivanovii, and L. welshimeri; innocua was recovered from the floor surrounding the raw product
Table 5) and L. monocytogenes, respectively. Locations that were posi- trailer and L. ivanovii was found in a trench drain (drain #2).
tive for Listeria spp. included drains, entry points, equipment legs, Subsequent intensified sampling rounds (n = 50/round) at Facility
floors, forklift tires, forklift traffic areas, and other locations (e.g., hand #1 expanded sampling into coolers, loading docks, transition areas, and
wash sink drain pipes, walls, temporary equipment, equipment outside, to areas outside the facility. Round C (March 2019) and round D (April
waste bins, raw product trailers, additional drain and floor locations, 2019) resulted in 16/50 (32%) and 15/50 (30%) samples testing po-
concrete seams on floors). L. monocytogenes was the most common sitive for Listeria spp., respectively. Positive sampling sites from round C
species found in PHP facilities (15/350; 4.3%), followed by L. wel- included drains (n = 6), the floor underneath raw product intake
shimeri (5/350; 1.4%), L. innocua (5/350; 1.4%), and L. ivanovii (1/350; (n = 1), step stools used by production employees (portable items,
0.3%). n = 2), a forklift tire (front left tire, n = 1), entry points (n = 2),
samples taken on outside surfaces (n = 3) and in the cooler (n = 1). L.
monocytogenes (n = 5) was recovered from all three drains in the
3.2. Serogroups of L. monocytogenes isolates from PHP facilities sorting/washing/packing area (drains #1–3). All three outside samples
(outside drain, concrete crack and tractor tire) were positive for L.
Seventy-five unique L. monocytogenes isolates recovered from PHP monocytogenes. L. monocytogenes was also found in a condensation pool
facilities belonged to two molecular serogroups: serogroup 1 (1/2a, 3a) near the entry point of one of the day-use coolers used to store bulk raw
and serogroup 4 (4 b, 4 d, 4e). Twenty-three isolates (31%) belonged to produce. L. welshimeri was recovered from one drain sample (drain #1),
serogroup 1 (lineage II) and 52 (69%) belonged to serogroup group 4 a step stool, and from an entry point, whereas L. innocua was isolated
(lineage I) (Table 6). from the outside drain. Positive sample sites during round D included
Environmental sampling at most facilities (4/5) resulted in the drains (n = 5), step stools (portable items, n = 2), entry points (n = 2),
isolation of a single serogroup. For example, all L. monocytogenes iso- outside sampling locations (n = 5), and the floor condensation in the
lates from Facilities #4 and #6 belonged to serogroup 1 (Table 6). L. raw produce storage cooler (n = 1). L. monocytogenes (n = 13) and L.
monocytogenes was also the only Listeria spp. recovered from environ- innocua (n = 4) were the two species recovered during round D. L.
mental samples in these two facilities. Facilities #4 and #6 are very monocytogenes was found in two drains at multiple locations (drain #1,
similar in the types of commodities that they process (mostly berries) drain #3), two different step stools, the floor near an entry point, and in
and their general processing methods. Two other PHP facilities (#2 and multiple samples collected outside Facility #1, including the three
#3) only had L. monocytogenes isolates that belonged to serogroup 4. L. positive sampling sites from round C. The cooler floor entry point
innocua was also isolated from environmental samples from these two condensation site was again positive for L. monocytogenes. L. innocua
facilities. These two facilities regularly work together and transport was recovered from one drain (drain #3), one step stool, and the floor
product back and forth between the two locations. One PHP facility, near two entry points.
Facility #1, is a packinghouse that handles and packs a large variety of In total, Listeria spp. were recovered from 44/150 samples (29%) for
fresh produce. The prevalence of Listeria spp. in this facility was quite all sampling rounds from Facility #1. Fig. 2 shows examples of sam-
high (> 26%) and included several Listeria spp. (L. innocua, L. ivanovii, pling sites separated into categories based on frequency of sampling site
L. monocytogenes, and L. welshimeri). Therefore, it is not surprising that testing positive for Listeria spp. in each sampling round: A) consistently
more than one serogroup of L. monocytogenes (1 and 4) was present in positive for Listeria spp. (positive all rounds), B) intermittently positive
the facility. L. monocytogenes isolates belonging to serogroup 4 were for Listeria spp. (positive in at least one round), C) consistently negative
more frequently isolated (40/52) than those belonging to serogroup 1 for Listeria spp. (negative all rounds). Sites that were consistently po-
(12/52). L. monocytogenes isolates from serogroups 2 (1/2c, 3c) and 3 sitive included all drains in the production area (4/4 rounds), an out-
(1/2 b, 3 b, 7) were not recovered from PHP facilities. door tractor tire (2/2 rounds), a wet floor location in a cooler near a
forklift entry point (2/2 rounds), and step stools (2/2 rounds). Listeria
3.3. Distribution of Listeria spp. in facility #1 spp. were commonly recovered from floors near entry points and on the
processing floors (3/4 rounds), whereas they were only occasionally
Overall results from four rounds (A, B, C, D) of environmental found on forklifts (1/4 rounds). Listeria spp. were not recovered from
sampling in Facility #1 are shown in Fig. 1. Sample collection during the outside of wood bins holding raw products, sides of processing
the initial sampling rounds (A and B) focused in the main receiving area equipment (0/4 rounds), other portable items (0/3 rounds) and the
where sorting, washing, and packing occur. During the first round of drain located in the transition area (drain #4) (Fig. 1). Species re-
sampling in Facility #1 (September 2018), only drain samples (3/20; covered for all rounds from Facility #1 included: L. monocytogenes
15%) were positive for Listeria spp. L. innocua isolates were recovered (n = 32), L. welshimeri (n = 8), L. innocua (n = 7), and L. ivanovii
from a single trench drain (drain #1) at two different locations and L. (n = 1).
monocytogenes was isolated from a second trench drain (drain #2).
During the second round of sampling (January 2019), the frequency of
isolating Listeria spp. increased to 10/30 (33%) samples. Sampling sites
5
J. John, et al. Food Microbiology 90 (2020) 103468
Fig. 1. Facility #1 map layout with all
sampling rounds identifying positive sites
for Listeria spp. and L. monocytogenes.
Sampling rounds were done at four different
time periods: September 2018 (A, n = 20),
January 2019 (B, n = 30), March 2019 (C,
n = 50), and April 2019 (D, n = 50).
Negative samples are highlighted in black
(A, B, C, D), positive samples for Listeria
spp. are highlighted in blue (A, B, C, D) and
positive samples for L. monocytogenes are
highlighted in red (A, B, C, D). Other Listeria
spp. may have also been recovered from red
sample sites (positive for L. monocytogenes).
Listeria spp. recovered included L. innocua,
L. ivanovii, L. monocytogenes and L. wel-
shimeri. (For interpretation of the references
to colour in this figure legend, the reader is
referred to the Web version of this article.)
3.4. Antibiograms of L. monocytogenes from facility #1 3.5. Listeria monocytogenes movement throughout facility #1
All tested L. monocytogenes isolates (n = 52) from Facility #1 were Further characterization of isolates, including serogrouping
sensitive to AMP, ERY, GEN, IMP, SXT, TET, and VAN and resistant to (Table 6) and AMR profiling (Fig. 3) suggested that three L. mono-
FOX and NAL (data not shown). In contrast, all isolates exhibited re- cytogenes strains (identical serogrouping and AMR profiles; designated
sistance or intermediate sensitivity to CLI and PEN (Fig. 3). NOV re- as C1, D1, and D2; Fig. 4) were isolated from multiple environmental
sistance was observed in three L. monocytogenes serogroup 4 isolates. samples. During the third round of testing, three L. monocytogenes ser-
These isolates also possessed resistance to CLI and PEN, indicating a low ogroup 4 isolates with identical AMR profiles (C1) were found on the
level of strains meeting the classification of multi-drug resistance production floor, on the floor near an entry point and in drain #3
(MDR). Notably, six and 16 isolates possessed intermediate sensitivity (Fig. 4; see photos in Fig. 2). In this facility, water is in near constant
to AMK and RIF, respectively (Fig. 3). use during produce handling, and the floor in this area is wet, especially
Fig. 2. Pictures of sampling sites from Facility #1
categorized by the frequency of detection of Listeria
spp. across all sampling rounds. Sites were separated
into three categories: (A) consistently positive for
Listeria spp. (positive all rounds); (B) intermittently
positive for Listeria spp. (positive at least one round);
or (C) consistently negative for Listeria spp. (negative
all rounds). Differences were assessed using chi-
square (p = 0.002) and Fisher's Exact (p = 0.00006).
6
J. John, et al. Food Microbiology 90 (2020) 103468
Fig. 3. Antimicrobial resistance patterns of
L. monocytogenes isolates (n = 40) from
Facility #1, separated by serogroups (1–4)
and strains with matching AMR patterns
based on nine antibiotics. The number (No.)
of isolates is in the white column with de-
termined and matching AMR profiles in the
next nine columns. Red “R” indicates re-
sistance, yellow “I” indicates intermediate
sensitivity and green “S” indicates sensi-
tivity to that antibiotic. (For interpretation
of the references to colour in this figure le-
gend, the reader is referred to the Web
version of this article.)
at the entry points and near drains. Furthermore, movement of em- is moved and stored throughout the facility. Employees stand on these
ployees, forklifts, or portable equipment through standing water or wet stools throughout the sorting, washing and packing processes and they
surfaces clearly demonstrates the potential dissemination of Listeria spp. are stored on the inside perimeter of the facility at various locations.
throughout the environment. It is likely that this L. monocytogenes strain This strain was also isolated from multiple locations in drain #1 and
(C1) entered Facility #1 from the side door (positive sample on floor from a single location in drain #3. D1 may have been tracked into the
near door with heavy foot traffic) and moved to the floor and drain on facility on the bottom of a production employee's shoe and deposited
the bottom of production employee shoes or by forklift tires. onto the step stool. Step stools and floors get washed down and cleaned
During the fourth round of testing, two sets of L. monocytogenes at the end of production and the strain could then be deposited into the
isolates with distinct AMR profiles (D1 and D2) were found in Facility drain. Alternatively, the drain could serve as the source for strain D1.
#1 (Fig. 4). The D1 strain was isolated from an employee step stool that Drains in this facility commonly get clogged with produce waste and
Fig. 4. Map of Listeria monocytogenes isolates suggesting routes of movement within Facility #1. Isolates with the same letter and number had the same serogroup and
antimicrobial resistance profile, suggesting potential relatedness.
7
J. John, et al. Food Microbiology 90 (2020) 103468
overflow onto the floor. Employee traffic in this area could result in outbreak, L. monocytogenes was recovered from the inside of wooden
Listeria spp. transfer to the bottom of worker footwear which may fa- storage bins (Angelo et al., 2017), though it is unknown if the bins were
cilitate transfer to the step stools and other areas of the facility. wet or dry. Overall, dry and wet locations across all sites were not in-
A third strain (D2) was isolated from an outside drain and a seam in cluded in the metadata, though this data could have contributed to
the concrete staging area in the middle of a produce delivery area at potential positive and negative comparisons.
this PHP facility. This strain was also found on the floor by a high-traffic Other locations, such as sides of processing equipment, were se-
side entry door and on the floor in the corner of a bulk produce cooler lected to asses potential cross contamination risks to food contact sur-
that accumulated moisture. This strain may have been brought into the faces. These sites are heavily cleaned and sanitized daily and may be a
facility by foot traffic or by a forklift and was likely spread to the cooler reason why we did not see any positives. Portable equipment (e.g.,
by extensive forklift traffic. It is possible that bins and pallets could wheels of scales, wheels of pallet jacks) were selected to investigate
facilitate transfer; however, we did not recovery any Listeria spp. on cross contamination throughout the facility. The wheels were generally
bins or pallets from this facility (0/8). made of hard plastic and typically wicked off any moisture, and they
were consistently dry upon sampling. Drains were targeted based on
4. Discussion previously reported high prevalence of Listeria spp. in drains in other
food production and retail environments (Berrang and Frank, 2012;
The prevalence of Listeria spp. in PHP facilities in the PNW differed Hoelzer et al., 2011; Kells and Gilmour, 2004; Tompkin, 2002). At
significantly by facility. Facilities fell into three categories, based on Facility #1, 68% (19/28) of drain samples were positive for Listeria
Listeria spp. prevalence: 1) no Listeria spp. detected, 2) low prevalence spp., with 63% (12/19) of drains containing L. monocytogenes. Results
(≤10% of environmental samples positive), or 3) high prevalence (26% from our study reaffirm the importance of preventing Listeria spp. co-
of environmental samples positive). Based on the diversity in facilities lonization of drains through scheduled cleaning and sanitization pro-
and the diversity of activities in PHP facilities, significant differences in grams. Special care should be taken to prevent drains from being
Listeria spp. prevalence could be expected; however, to date, this is the clogged and overflowing onto the floor.
first study to strategically sample similar areas in categorically different Notably, L. monocytogenes was recovered from the floor near the
facilities and demonstrate differences in prevalence in these PHP fa- entry way of the bulk produce storage cooler sample site (floor with
cilities. A previous three-year study by Leong et al. (2017) reported that moisture) in rounds C and D (two separate sampling dates) in Facility
the prevalence of L. monocytogenes varied across facilities from multiple #1. This could point to L. monocytogenes persistence and adaptation at
food industries, including five vegetable processing facilities. Certain this location and a potential niche for Listeria spp. Upon the second
vegetable processing facilities maintained a prevalence of 0% while one positive in the last round, Facility #1 management proceeded with
facility had a prevalence of 30% during a single sampling year. Vege- corrective actions to frequently clean this location and eliminate the
table processing facilities had the highest environmental prevalence for buildup of moisture. Serogrouping and AMR profile data on this isolate
L. monocytogenes as compared to the meat, seafood and dairy processing suggested traffic patterns as the mechanism for contamination of this
facilities (Leong et al., 2017). A recent study by Tan et al. (2019) re- site; however, additional genetic characterization would be necessary
ported the prevalence of L. monocytogenes in three tree fruit processing to confirm that these isolates are persistent. Production employee shoes
facilities, with one facility having extremely high prevalence (39/39; were not sampled; however, positive samples on floors with heavy foot
100%). High prevalence was attributed to several factors, including traffic (main door entrances, step stools) suggested that movement of
lack of a proper drainage systems, cracks in floors and poor cleaning workers and equipment in this facility could disseminate Listeria spp.
and sanitation practices (Tan et al., 2019). throughout the facility. It is also possible that there is an environmental
Facility #1 had a significantly higher prevalence of Listeria spp. in niche for L. monocytogenes in the cooler. Targeted sampling in the cooler
the environment than all other PHP facilities. This facility is an enclosed area would help to identify the source and movement of these strains in
packinghouse that sorts, cleans, and stores a diversity of crops. Facility the facility. Only a few contamination scenarios were addressed in this
#1 was the only participating PHP facility that was subject to the PSR study based on the available strain relatedness data. There is some
and not subject to PCHF. The current regulatory landscape for this type evidence from these scenarios for a connection between high traffic
of PHP facility does not require, nor encourage, the establishment of a areas and the movement of foot traffic and forklift traffic in raw pro-
pathogen environmental monitoring program; therefore, Facility #1 cessing areas. Muhterem-Uyar et al. (2015) observed three L. mono-
had no previous data on the prevalence of Listeria spp. in their opera- cytogenes contamination scenarios in 12 meat and dairy facilities across
tion. Based on the results from this study, the food safety personnel (one Europe that relate to our findings. From 2242 environmental samples
person) at Facility #1 began testing environmental samples to verify this study found: (i) sporadic contamination associated with raw ma-
the efficacy of their cleaning and sanitation programs and they intend terial reception and hygienic areas; (ii) hotspots in hygienic processing
to develop a Pathogen Environmental Monitoring Program for Listeria areas; and (iii) widespread contamination throughout the entire facility
spp. Close collaboration with the PHP facilities facilitated their ability (Muhterem-Uyar et al., 2015). Particularly, scenario (i) described by
to understand parts of their processing environment that may require this study corroborates our findings, observing that a contamination
additional focus for cleaning and sanitation programs. from the outside environment was due to the lack of hygiene barriers in
Sampling locations across PHP facilities were selected to evaluate raw material reception areas (Muhterem-Uyar et al., 2015). No hygienic
sites considered to be at high risk for contamination with Listeria spp. barriers are present in Facility #1 and there is near constant exposure to
(i.e., transition floors indoor to outdoor, raw product entry, outside the outside environment. This is a common and unavoidable situation
sites and handling areas) (Carpentier and Cerf, 2011; Muhterem-Uyar in packinghouse facilities. The current study demonstrated a high pre-
et al., 2015; Tompkin, 2002). Through preliminary interviews we valence of Listeria spp. entering the facility as product is received and
learned that wood bins and pallets were reused, occasionally trans- moved. Therefore, focused efforts should be on cleaning and sanitation
ferred and stored outside the facility, and are rarely cleaned and sani- programs that will prevent cross-contamination from crop to crop or
tized. The bins and pallets were sampled on the outside, which were from day to day.
always dry. Keeping equipment and the production environment dry is Four species of Listeria were isolated from Facility #1: L. innocua, L.
a key component in Listeria spp. control (Tompkin, 2002), and could be ivanovii, L. monocytogenes, and L. welshimeri. These species are the most
a contributing factor why we did not see any positives on bins and commonly recovered species from various food and food-associated
pallets, rather than the bins being wooden. L. monocytogenes was re- environments; however, L. ivanovii is less frequently isolated (El-
covered from two wood panels outside the facility (i.e., ground in front Shenawy, 1998; Gebretsadik et al., 2011; Heisick et al., 1989;
of outside bathrooms) that were wet. During the 2017 caramel apple Kovacevic et al., 2009; Wilson, 1995). Facility #1 handles a wide
8
J. John, et al. Food Microbiology 90 (2020) 103468
variety of fresh produce. On sampling days, parsnips, cabbage, and References
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interests or personal relationships that could have appeared to influ-
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Risk factors associated with Salmonella and Listeria monocytogenes contamination of
10