Food Safety Testing

Testing Environmental Sponge Samples
for Next-Day Listeria Species Results

Jennifer Pelowitz, Wendy Lauer, Joshua Whitworth
Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Dr, Hercules, CA 94547 USA

Abstract

A global food and agriculture company partnered with Bio-Rad Laboratories, Inc. to evaluate methods
to detect high levels of Listeria spp. in environmental sponge samples with a target 6–12 hr sample
enrichment period to obtain quantitative PCR (qPCR) results in one day. Current methods typically haveincubation times of 24 hr. In previous studies, we identified the minimum incubation period required todetect multiple strains of Listeria at an inoculum of 30–50 colony forming units (CFU) with theiQ-Check™ Listeria spp. PCR Detection Kit method with and without preheating the enrichment media(data not shown). Initial testing showed that with prewarmed media, positive test results for Listeria spp.
can be obtained following a 4–5 hr incubation period with the iQ-Check Standard II Protocol. To further
challenge the method and simulate real-world conditions, environmental sponges were obtained froma manufacturing production plant containing normal flora for subsequent inoculation with Listeria in thelaboratory. A new highly nutritive enrichment medium, Listeria Special Broth II (LSB II), was tested andproved to facilitate the reduced enrichment time.
Introduction
Listeria spp. are gram-positive, rod-shaped bacteria that are motileby flagella. They are hardy and survive many harsh environmental
conditions such as low temperature, low pH, freezing, drying, andheating. These environmental organisms are found in soil andwater, on vegetation, and on surfaces in food processing facilities.
The genus Listeria is often used as an indicator of improper
sanitation procedures for equipment and possible contamination
with pathogenic Listeria monocytogenes. Listeria ssp. have beenfound throughout the environment of processing plants on various
surfaces including equipment, tools, vents, floors, and foodtransport racks, making control of the bacteria on environmental
surfaces a critical control point. The timely acquisition of Listeria
environmental results allows for corrective actions to be taken, apivotal factor in bolstering food safety protocols and protecting
the integrity of the food supply chain.

Bulletin 3619

Methods
Environmental samples collected using polyurethane environmental
sponges with HiCap Neutralizing Broth (World Bioproducts,catalog #EZ-10HC-PUR) were received from the production plant
and randomly assigned a number, inoculum, and incubation time
(Table 1). Samples were directly inoculated with a target
30–50 CFU of either Listeria monocytogenes, ATCC 13932(actual CFU, 43) or Listeria grayi, ATCC 25401 (actual CFU, 49),
and 100–1,000 CFU of competing organism Staphylococcus
aureus, ATCC 6538 (actual CFU, 482). Sponges were enrichedwith 60 ml Listeria Special Broth II (LSB II, Bio-Rad Laboratories,Inc., #12017378) prewarmed to 37°C and incubated for 4, 5, or
6 hr. After incubation, samples were processed with the Standard
II Protocol (document #10000167777), and the resulting DNA
samples were tested in triplicate using the iQ-Check Listeria spp.
Detection PCR Kit (Bio-Rad, #3578113) and the FAST thermal
protocol on a CFX96 Touch Deep Well Real-Time PCR Detection
System (Bio-Rad, #3600037).

Testing Environmental Sponge Samples for Next-Day Listeria Species Results

Table 1. Sample log and experimental setup.
SamplePlant Location

Inoculum Target Organism

Inoculum Competing Organism

Enrichment Time, hr

1

Bottom of belt

L. grayi

S. aureus

42

Hose #1

L. grayi

S. aureus

43

Rails under belt

L. grayi

S. aureus

44

Ceiling exhaust by dicer

L. monocytogenes

S. aureus

45

Inedible barrel wheel mounts

L. monocytogenes

S. aureus

46Floor under shaker table

L. monocytogenes

S. aureus

47

Stairs to leveling belt

L. grayi

S. aureus

58

Hose #2

L. grayi

S. aureus

59Metal detector control panel

L. grayi

S. aureus

510

Legs of metal detector #2

L. monocytogenes

S. aureus

511Dicer framework

L. monocytogenes

S. aureus

512

Blade cart

L. monocytogenes

S. aureus

513

Drain unclogging tool

L. grayi

S. aureus

614

Incline belt to shaker table

L. grayi

S. aureus

615

Legs of metal detector #1

L. grayi

S. aureus

616

Drain

L. monocytogenes

S. aureus

617

Legs at chiller exit

L. monocytogenes

S. aureus

618

Chiller door

L. monocytogenes

S. aureus

619

Framework of metal detector #2L. grayi—520

Crack in floor behind chiller

L. monocytogenes—5The sponges were returned to the incubator for an additional
16–18 hr, after which they were streaked to Modified Oxford
Medium (MOX, Hardy Diagnostics, #G46) and RAPID'L.mono
Agar Plates (RLM, Bio-Rad, #3563694) and incubated at 35± 2°C for 24–28 hr. Typical colonies were confirmed with qPCR.
Simultaneously, 0.1 ml of the primary enrichment was added to 10 ml
4-morpholinepropanesulfonic acid–buffered Listeria enrichment
broth (MOPS-BLEB) prepared from MOPS, Free Acid, MolecularBiology Grade (MilliporeSigma, #475922-100GM), MOPS-Na ≥99%, High Purity (VWR International, LLC., #E413-250G), andCriterion Listeria Enrichment Broth Base, Dehydrated Culture
Media (Hardy Diagnostics, #C9301) and incubated at
35 ± 2°C for 18–24 hr. A secondary qPCR analysis was performed
with the MOPS-BLEB culture. To remove PCR inhibition, a 1:10
dilution was needed for qPCR on MOPS-BLEB enrichments.

Primary enrichments for sponges that did not show growth weretested with Mquant Quaternary Ammonium Compounds TestStrips (MilliporeSigma, #1.17920.0001).
Results
4-Hr Enrichments

The combination of prewarmed LSB II and DNA sample
preparation using the Standard II Protocol following incubation for
4 hr gave inconsistent results. Some samples that tested negative
after 4 hr had positive results with a secondary enrichment (Table2). Samples that were fractionally positive at 4 hr and negative
after secondary enrichment were assumed to be the result of theinoculum dying due to quaternary ammonia on the sponges. These
unconfirmed positive results could potentially be removed by using
iQ-Check Free DNA Removal Solution (FDRS, Bio-Rad, #3594970)
if the cause was free DNA in the sample.

Table 2. Results following a 4-hr enrichment. Replicates show fractional recovery and inconsistencies with secondary enrichment results.
Same-Day qPCR Results
Time
Point Sample Organisms

4 hr

Cq 1

Cq 2

Cq 3

Avg

Secondary Enrichment qPCR Results

Rep 1 Rep 2 Rep 3 Cq 1

Cq 2

Cq 3

Avg

Confirmation Results

RLM
Rep 1 Rep 2 Rep 3 AgarPCR
MOX Colony
Agar Confirm

Quats,
mg/L

137.84 35.96 37.39 37.06+++25.82 26.29 25.84 25.98+*+++++2

L. grayi, S. aureus 34.88 35.00 35.40 35.09+++21.47 21.68 21.82 21.66+*+++++NA

340.84 ND

40.84

+––

27.58 27.46 27.22 27.42+++––+–30

435.81 ND
38.10 36.96
L. monocytogenes,
36.85 40.01 36.38 37.75
S. aureus
39.39 40.53 ND
39.96

+–+

18.66 18.73 19.04 18.81++++++

NA+++

19.06 19.00 18.97 19.01++++++NA++–ND–––––––30

56ND

NDNDNDNA* RLM positive for Listeria ssp. other than L. monocytogenes.
Cq, quantification cycle; MOX, Modified Oxford Medium; NA, not tested; ND, not detected; Quats, quaternary ammonium compounds; Rep, replicate samples;
RLM, RAPID'L. mono Agar Plates.

© 2024 Bio-Rad Laboratories, Inc.

2

Bulletin 3619

Testing Environmental Sponge Samples for Next-Day Listeria Species Results

Table 3. Results following 5- and 6-hr enrichements. Replicates show consistent results compared to secondary enrichments.
Same-Day qPCR Results
TimePointSample Organisms
78L. grayi, S.aureus

95 hr1910L. grayi

Rep 1 Rep 2 Rep 3 Cq 1

Cq 2Cq 3

Avg

Confirmation Results

PCR
RLM MOX Colony Quats,
Rep 1 Rep 2 Rep 3 Agar Agar Confirm mg/L

34.79 35.46 34.71 34.99+++22.36 22.19 22.41 22.32++++*++<30

34.15 34.76 35.39 34.77+++

21.89 21.64 22.13 21.89++++*++

NA

36.52 34.75 35.53 35.60+++

21.74 21.76 21.54 21.68++++*++

NA

34.13 36.17 37.92 36.07+++19.46 19.66 19.90 19.67++++*++NA35.85 35.03 35.69 35.52+++

19.13 19.14 19.60 19.29++++++NA+++

19.62 19.37 19.44 19.48++++++NA+++

19.11 19.23 19.12 19.15++++++

<30

12

L. monocytogenes,
37.78 36.91 36.87 37.19
S. aureus
36.28 37.08 34.90 36.09

20

L. monocytogenes ND

1139.21

–+–

ND

–––––

–

~100

34.59 34.86 34.02 34.49+++

21.30 21.48 21.39 21.39++++++NA

37.68 35.87 35.72 36.42+++

22.46 22.42 22.53 22.47++++++NA1534.20 33.79 35.05 34.35+++22.81 22.80 22.92 22.84++++++NA

16ND

––+ND

––––––~75+++

19.46 19.15 19.56 19.39++++++NA+++

19.03 19.92 19.49 19.48++++++

NA13
146 hr

Cq 1 Cq 2 Cq 3 Avg

Secondary Enrichment qPCR Results

1718

L. grayi, S. aureus

39.21 ND

ND

38.78 38.78

L. monocytogenes,
35.20 35.05 34.62 34.96
S. aureus
33.92 33.61 33.76 33.76

ND

ND

ND

ND

ND

ND

* RLM positive for Listeria ssp. other than L. monocytogenes.
Cq, quantification cycle; MOX, Modified Oxford Medium; NA, not tested; ND, not detected; Quats, quaternary ammonium compounds; Rep, replicate samples;
RLM, RAPID'L. mono Agar Plates.

Conclusions
A 4-hr incubation period for the inoculated sponges gaveinconsistent results; however, a minimum incubation time of 5 hrgave consistent positive results except in the presence of quaternaryammonia. Additional work can be done to treat unconfirmed positivesamples with FDRS and retest to see if the result was due to free
DNA in the sample. LSB II was designed to be a highly nutritiveenrichment media, particularly for injured Listeria cells, allowing
for a decreased sample enrichment time. Obtaining Listeria spp.
environmental results 20 hours earlier than currently available
methods is paramount to ensure food safety. Rapid identification ofpotential sources of contamination enables timely corrective actions,preventing the possible spread of Listeria in the food processingenvironment. This proactive approach safeguards consumers
from the risk of consuming contaminated products and minimizes
economic and reputational consequences for food manufacturers.

5- and 6-Hr Enrichments

In contrast to the time point at 4 hr, enrichments ≥5 hr withprewarmed LSB II followed by DNA sample preparation using
the Standard II Protocol gave consistent results when compared
to secondary enrichment (Table 3). Samples that were fractionally
positive and negative after secondary enrichment were assumed tobe the result of the inoculum dying due to quaternary ammonia on
the sponges.
Unconfirmed Positive and Negative Results
Samples that produced negative and unconfirmed positive qPCR
results after inoculation (20 and 16) were tested with Mquant
Quaternary Ammonium Test Strips to determine whether abactericide cleaner was picked up and not neutralized whenswabbing with the environmental sponges. In all cases, sponge
enrichments tested positive for 30–100 mg/L of quaternaryammonia. For comparison, two random samples (7 and 12) that
tested positive after both primary and secondary enrichments werealso analyzed, producing negative results for quaternary ammonia.

Visit bio-rad.com/iQCheck to learn more.
BIO-RAD is a trademark of Bio-Rad Laboratories, Inc. IQ-CHECK is a trademark of
Bio-Rad Europe GmbH in certain jurisdictions. All trademarks used herein are theproperty of their respective owner. © 2024 Bio-Rad Laboratories, Inc.

© 2024 Bio-Rad Laboratories, Inc.

3

Bulletin 3619

Testing Environmental Sponge Samples for Next-Day Listeria Species Results

Bio-RadLaboratories, Inc.
Website bio-rad.com USA 1 800 424 6723 Australia 61 2 9914 2800 Austria 00 800 00 24 67 23 Belgium 00 800 00 24 67 23 Brazil 4003 0399
Canada 1 905 364 3435 China 86 21 6169 8500 Czech Republic 00 800 00 24 67 23 Denmark 00 800 00 24 67 23 Finland 00 800 00 24 67 23France 00 800 00 24 67 23 Germany 00 800 00 24 67 23 Hong Kong 852 2789 3300 Hungary 00 800 00 24 67 23 India 91 124 4029300 Israel 0 3 9636050
Italy 00 800 00 24 67 23 Japan 81 3 6361 7000 Korea 82 080 007 7373 Luxembourg 00 800 00 24 67 23 Mexico 52 555 488 7670
The Netherlands 00 800 00 24 67 23 New Zealand 64 9 415 2280 Norway 00 800 00 24 67 23 Poland 00 800 00 24 67 23 Portugal 00 800 00 24 67 23Russian Federation 00 800 00 24 67 23 Singapore 65 6415 3188 South Africa 00 800 00 24 67 23 Spain 00 800 00 24 67 23 Sweden 00 800 00 24 67 23
Switzerland 00 800 00 24 67 23 Taiwan 886 2 2578 7189 Thailand 66 2 651 8311 United Arab Emirates 36 1 459 6150 United Kingdom 00 800 00 24 67 23

Life ScienceGroup

Bulletin 3619 Ver A

US/EG24-0841

0324

Sig 0124