Food Protection Trends - September/October 2020 - 322

only) were used to collect data at another nine Iowa retail
delicatessen establishments. Similar results to the first pilot
occurred with test kits producing false-positive results.
On the basis of these results, the decision was made to use
traditional sponge stick sampling methodology for the final
sampling assignment.
Final collection
There were 57 small and large retail deli operations in Iowa
that were randomly selected for the final data collection.
The selection was based on a quota sampling technique with
approximately 75% (n = 43) of selected operations being small
and the remaining approximately 25% (n = 14) being large
operations. Sampling at more small operations was intentional,
given the higher frequency of violations in small delis. All data
were collected during routine health inspections.
Microbial sample collection
Three trained individuals visited and collected microbial
samples by using a traditional gloved sponge stick technique.
Inspectors were trained in aseptic sample collection
techniques, which is the standard method for all FDA
consumer safety officers during sampling assignments
(48). Each sampling required the inspector to collect five
to seven samples from clean food contact areas used for
RTE delicatessen meat preparation or storage. Sampling
site examples were as follows: slicer, meat scale, cutting
board, walk-in cooler racks, and deli meat cases. Inspectors
labeled each sample bag with the location, sample number,
and sample lot number. Information from each sample was
captured on a sample collection form with location, sample
zone, sample number, and sample lot code. The operator was
instructed to reclean any sampled equipment. All samples
were shipped the same day in soft-sided coolers to the State
Hygienic Laboratory for analysis with the designated chain
of custody forms.
Microbiological analyses
For the final data collection activity, World Bioproducts
EZ Reach™ sterile polyurethane sponges that contained
10 mL of Dey-Engley neutralizing broth (EZ-10DE-PUR,
World Bioproducts, Bothell, WA) were used for collection
and sent to the State Hygienic Laboratory for Listeria species
testing and culture confirmation. The microbiological
procedures used for the environmental sample processing
were from the FDA Bacteriological Analytical Manual (49)
and Hygiena BAX System Real-Time PCR Assay for Genus
Listeria (D15131113, Hygiena, LLC). Upon receipt in the
laboratory, the sponges were homogenized (vigorously
hand massaged) with prewarmed University of Vermont
media (modified Listeria enrichment broth; Difco, BD,
Sparks, MD) broth and incubated at 30°C for 20 to 26 h.
After primary incubation, 0.1 mL of primary enrichment
was transferred into 10 mL of morpholinepropanesulfonic

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Food Protection Trends September/October

acid-buffered Listeria enrichment broth (BD Bacto; BD)
with antibiotics (0.5% acriflavine, 0.5% nalidixic acid, and
1% cycloheximide solutions, Sigma-Aldrich Corporation,
St. Louis, MO) and incubated at 35°C for 18 to 24 h.
Real-Time PCR by using Hygiena BAX System Real-Time
PCR Assay for Genus Listeria (D15131113, Hygiena,
LLC) was performed first. If the PCR test was positive,
aliquots from morpholinepropanesulfonic acid-buffered
Listeria enrichment broth were transferred to two agar
media: CHROMagar Listeria (CHROMagar, Paris, France)
and modified Oxford Listeria selective agar (Difco, BD)
containing modified Oxford antimicrobic supplement (Difco,
BD). Suspect Listeria colonies were isolated on sheep blood
agar plates (Difco, BD) and the Listeria genus and species
were confirmed by using matrix-assisted laser desorption-
ionization time of flight (Daltonics Microflex LT, Bruker
Corporation, Boston, MA) mass spectrometry.
All five Listeria monocytogenes isolates recovered for this
project were submitted for whole-genome sequencing at
the State Hygienic Lab (Table 1). Sequencing material and
methods are given in the following.
DNA isolation
DNA isolation was performed on a QIAcube (Qiagen,
Hilden, Germany) instrument by using the QIAamp DNA
Mini Kit (catalog #51306, Qiagen) according to the manufacturer's protocol. Prior to loading bacterial cells on the
instrument, an enzymatic digestion was performed by using
lysozyme (20 mg/mL; catalog #L4919, Millipore Sigma, St.
Louis, MO) and RNase A (11 ng/μL final concentration;
catalog #19101, Qiagen). DNA was eluted in 100 L of 10
mM Tris HCl, pH 8. DNA concentrations were measured
on a Qubit 3.0 Fluorometer (Thermo Fisher Scientific Inc.,
Waltham, MA) by using the Qubit dsDNA BR Assay Kit
(catalog #Q32853, Thermo Fisher Scientific Inc.).
Library preparation and sequencing
Libraries were prepared by using the Illumina Nextera
XT DNA Library Prep Kit (catalog #FC-131-1096) and
Nextera XT Index Kit v2 (catalog #FC-131-2003, Illumina
Inc., San Diego, CA) following the FDA Center for Food
Safety and Applied Nutrition protocol "Nextera XT Library
Preparation for Sequencing on a MiSeq" (29). Libraries
quality were inspected by using the Qubit dsDNA HS
Assay Kit (catalog #Q32854, Thermo Fisher Scientific Inc.,
Waltham, MA) and the Agilent Bioanalyzer High Sensitivity DNA kit (catalog #5067-4626, Agilent Technologies Inc.,
Waldbronn, Germany). Libraries for Listeria were pooled
together with other bacterial libraries to reach a total of 160
Mb per run and sequenced on an Illumina MiSeq instrument (Illumina Inc.) for 2 × 250 cycles by using a MiSeq
Reagent Kit v3 (catalog #MS-102-3003, Illumina Inc.).

Food Protection Trends - September/October 2020

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