Controlling Salmonella and L. monocytogenes
with a comprehensive farm-to-table approach
Martin Wiedmann
Department of Food Science
Cornell University, Ithaca, NY
E-mail: mw16@cornell.edu
Phone: 607-254-2838
Cornell University
Outline
• Introduction & overview of foodborne pathogens of
concern
• Use of DNA fingerprinting methods to detect disease
outbreaks and analyze contamination patterns
• Key pathogens
– Listeria monocytogenes
– Salmonella
• Developments in molecular detection
• Conclusions
Microbial foodborne diseases
• Latest 2011 CDC study estimates 47.8 million cases of
gastrointestinal illnesses ; 9.4 million due to known
and 38.4 million due to unknown pathogens)
– 127,000 serious illnesses resulting in hospitalizations;
56,000 due to known and 71,000 due to unknown
pathogens
– 3,037 deaths (range: 1,492–4,983); 1,351 due to known
and 1,686 due to unknown pathogens
What pathogens to worry about
• Know the foods you produce (raw materials; properties such as
water activity, pH; consumer use)
• Identify likely pathogens present as well as outbreaks linked to a
given food
• Understand pathogen prevalence, levels as well as growth or
reduction that occurs during processing and storage
• Understand both public health risk (risk that food causes disease
case or outbreak) as well as regulatory and business risk (risk of
recall in absence of adverse public health consequences)
• Re-evaluate risk regularly and when new data become available
Global Food Supply
Cocoa PowderSwitzerland
Roasted
Espresso
BeansColumbia
Mascarpone
Cheese and
LadyfingersItaly
Vanilla BeansMadagascar
ChocolateBelgium
KahluaMexico
Mint Leaf –
United States
CreamUnited States
Copyright  Yiannas
14
Transmission of foodborne zoonotic diseases
Animal feed/environment/protozoans
Food animals
Manure
Animal derived food products
Water
Plant derived raw products
Food Processing Plants
RTE Foods
Humans
15
Outline
• Introduction & overview of foodborne pathogens of
concern
• Use of DNA fingerprinting methods to detect disease
outbreaks and analyze contamination patterns
• Key pathogens
– Listeria monocytogenes
– Salmonella
• Developments in molecular detection
• Conclusions
Strain differentiation (subtyping/fingerprinting)
• Tools which allow sensitive differentiation of bacterial
subtypes
– Detection of contamination sources
• Strain differentiation methods commonly applied
include serotyping, ribotyping, Pulsed Field Gel
Electrophoresis (PFGE)
• These methods are used to detect foodborne disease
outbreak and identify pathogen sources throughout the
food chain
Examples of L. monocytogenes ribotypes
18
Examples of different PFGE patterns
M
M
M
19
DNA sequencing-based
subtyping
88
2 289
j2 -04 5
75
j1 -03 8
92
L99
j2 -06 8
92
j2 -00 3
1 040 3S
92
91
92
j1 -04 7
c 2-00 6
n 1 -064
75
c 2-00 8
92
d d 68 0
c 2-01 1
n 1 -067
89
92
Isolate 1 AACATGCAGACTGACGATTCGACGTAGGCTAGACGTTGACTG
Isolate 2 AACATGCAGACTGACGATTCGTCGTAGGCTAGACGTTGACTG
Isolate 3 AACATGCAGACTGACGATTCGACGTAGGCTAGACGTTGACTG
Isolate 4 AACATGCATACTGACGATTCGACGAAGGCTAGACGTTGACTG
Case study – human listeriosis
outbreak
Human listeriosis cases in NYS: 1/97-10/98
8
7
6
5
4
3
2
1
O
ct
Au
g
Ju
n
M
ar
Ja
n
N
ov
S
ep
Ju
l
M
ay
M
ar
Ja
n
0
Ribotyping results - Nov 8, 9 pm
Ribotyping results - Nov 8, 12 pm
Epidemic curve for 1/97 - 2/99 in NYS
8
1044A
Other Ribotypes
7
6
5
4
3
2
1
Fe
b
D
ec
O
ct
Au
g
Ju
n
M
ar
Ja
n
No
v
Se
p
Ju
l
M
ay
M
ar
Ja
n
0
Similarity Search Results
Conclusions
• 101 human cases and 21 deaths in 22 US states linked
to infection by the same sub-type of Listeria
monocytogenes
• Outbreak traced back to a single specific plant in
Michigan
PulseNet Canada
PulseNet
Europe
PulseNet
USA
PulseNet
Middle East
PulseNet
Latin America
& Caribbean
PulseNet
Asia
Pacific
Possibilities for
international traceback –
a hypothetical example
PulseNet Canada
PulseNet
Europe
Food isolate, deposited into
PulseNet 06/2010
Human case, Canada 05/2011
PulseNet
USA
PulseNet
Middle East
PulseNet
Asia
Pacific
PulseNet
Latin America
& Caribbean
Human case, US 05/2011
L. monocytogenes ecology and
contamination patterns in seafood
processing plants
• Environmental Listeria contamination as
significant problem in the food industry
• Controlling environmental L. monocytogenes
contamination in food plants is key to better
control (“Seek and destroy”)
DNA fingerprinting can identify
persistence in plants
Sample
VISIT 1
VISIT 2
VISIT 3
Ribotype
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
1039C
1039C
1039C
1039C
1039C
1039C
1039C
1039C
1044A
1044A
1044A
1045
1045
1053
1062
1039C
1039C
1039C
1039C
1039C
1039C
1039C
1039C
1044A
1044A
1062
Sample Source
Sample Source
RiboPrint® Pattern
(E) Floor drain, raw materials area
(E) Floor drain, hallway to finished area
(IP) Troll Red King Salmon, in brine, head area
(IP) Troll Red King Salmon, in brine, belly area
(IP) Brine, Troll Red King Salmon
(IP) Faroe Island Salmon, in brine, head area
(F) Smoked Sable
(F) Cold-Smoked Norwegian Salmon
(E) Floor drain, brining cold room 1
(R) Raw Troll Red King Salmon, head area
(IP) Brine, Faroe Island Salmon
(R) Raw Troll Red King Salmon, belly area
(IP) Faroe Island Salmon, in brine, head area
(IP) Norwegian Salmon, in brine
(E) Floor drain #1, raw materials preparation
(E) Floor drain #1, raw materials preparation
(E) Floor drain, brining cold room 1
(E) Floor drain #2, raw materials preparation
(E) Floor drain #2, raw materials receiving
(E) Floor drain, finished product area
(E) Floor drain, hallway to finished area
(IP) Brine, Troll Red King Salmon
(F) Smoked Sable
(IP) Sable, in brine
(IP) Brine, Faroe Island Salmon
(IP) Brine, Norwegian Salmon
L. monocytogenes persistence in plants
Samples
Plant B
Plant C
Plant D
n=129
n=173
n=229
Ribotype
P-value
% Prevalence
1039C
0.0
0.0
10.0
0.0000
1042B
0.8
1.2
0.4
0.8221
1042C
6.2
0.6
0.4
0.0003
1044A
0.0
2.3
3.1
0.1494
1045
5.4
0.0
0.9
0.0006
1046B
0.0
2.3
0.0
0.0144
1053
0.0
0.6
1.7
0.2686
1062
0.8
0.6
2.6
0.1822
33
2000 US outbreak - Environmental
persistence of L. monocytogenes?
• 1988: one human listeriosis case linked to hot dogs produced
by plant X
• 2000: 29 human listeriosis cases linked to sliced turkey meats
from plant X
Summary - Persistent L. monocytogenes
in food processing plants
• Persistent environmental contamination has been reported
in almost all types of food processing plants, including RTE
seafood plants (> 10 years), dairy plants; RTE meat plants
(>12 years); poultry processing plants etc.
• A number of listeriosis outbreaks have been linked to
persistent L. monocytogenes contamination in source plants
• Industry has adapted the “Seek and Destroy” strategy to
address this issue
L. monocytogenes in retail environments
• Subtyped 98 food and 40 environmental L. monocytogenes
isolated from 50 supermarkets in New York State between
1997 and 2002
• 16 supermarkets showed evidence for persistence of one or
more specific L. monocytogenes strains as indicated by
isolation of the same EcoRI ribotype from food and/or
environmental samples collected in a given establishment on
different days
• 17 ribotypes were found among human clinical isolates as
well as among food and environmental isolates
Sauders et al. 2004. J. Food. Prot. 67: 1417–1428
Persistence in a retail store - example
STORE 7
Food Contact Sites
Slicer
Deli case
Deli case near raw meat
Deli case trays
3-basin sink interior
1-basin sink interior
Cold room rack
Cutting board
Rewrap table
Counter
Non-food contact sites
3-basin sink exterior
Floor/wall junction (3-basin)
1-basin sink exterior
Floor/wall junction (1-basin)
Deli drain
Floor adjacent to drain
Deli floor
Cold room floor
Cold room wall
Cold room drain
Standing water
Squeegee
Cart Wheel
Hose
Trash can
Transfer Points
Slicer knob
Case handle
Scale
April
May
June
July
August
September
October
November
December
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
CU-57,267
CU-258,69
NT
-
-
-
NT
-
CU-294,321
NT
-
-
NT
CU-258,69
NT
NT
NT
NT
NT
CU-258,69
NT
NT
NT
NT
-
NT
CU-258,69
NT
NT
NT
CU-258,69
NT
NT
NT
NT
NT
NT
-
NT
CU-258,69
NT
NT
NT
CU-258,69
NT
NT
NT
NT
NT
CU-258,69
NT
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
NT
CU-258,69
CU-258,69
-
CU-258,69
CU-258,333
CU-258,69
CU-295,329
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-8,96
-
LM
LM
LM
CU-258,69
CU-258,69
CU-258,69
NT
CU-258,69
-
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
NT
CU-258,69
-
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
CU-258,69
-
NT
NT
NT
-
-
-
-
-
-
38
Outline
• Introduction & overview of foodborne pathogens of
concern
• Use of DNA fingerprinting methods to detect disease
outbreaks and analyze contamination patterns
• Key pathogens
– Listeria monocytogenes
– Salmonella
• Developments in molecular detection
• Conclusions
Listeria monocytogenes
Gram-positive rod
•Commonly found in the
environment
• Unique ability to survive and
even grow in extreme
environments…
•
o
o
Growth at temperatures: –0.4 C to 50 C
Survival under acid conditions (pH 4.5 )
Survival in solutions of 10-15 % NaCl
Listeria monocytogenes
• Causes foodborne disease
• Causes septicemia, abortion and encephalitis in humans and more than
40 animal species, but is also common in environment
• Potentially long incubation period (7-60 days)
• High infectious dose: at 1 x 1010 cfu/serving, the dose-response model
predicts a median death rate of 1 in 667 servings for pregnancy
associated/neonatal listeriosis
• Human listeriosis can occur as epidemic and sporadic cases
• Affects predominantly elderly and immunocompromised people,
pregnant women and newborns.
• Approx. 1,300 human cases/year and 255 deaths/year in the US
• Common in certain/many environments
L. monocytogenes prevalence
• Pristine environments: 1.3% (n=900)
• Urban environments: 7.3% (n=900)
• Ruminant farms
–
–
–
–
Bovine farms with listeriosis cases: 24.3% (n=616)
Bovine farms without listeriosis cases: 20.1% (n=643)
Small ruminant farms with listeriosis: 32.9% (n=322)
Small ruminant farms without listeriosis: 5.9% (n=475)
• Raw foods
• Food processing environments: from <0.1% to 30% or more
• Ready-To-Eat foods (US data): 0.17 – 4.7 % (Gombas et al., 2004)
Control of Listeria monocytogenes
• Predominant concern are Ready-To-Eat (RTE) foods that (i) permit L.
monocytogenes growth and (ii) are stored for prolonged time
– Examples include RTE meats, smoked seafood, certain soft cheeses (brie,
blue cheese, other cheeses with high water activity and pH)
– Emerging concern are produce items (cantaloupe outbreak in US, recalls
of sliced apples)
• Control strategies need to focus on preventing post kill step recontamination of products (at plants as well as at retail)
– Sanitary equipment design
– Environmental testing
– Appropriately designed and implemented SSOPs (sanitation standard
operating procedures)
• Control of L. monocytogenes on products that do not have a kill step (i.e.,
fresh produce) is a considerable challenge
Selected Foodborne Pathogens
in the Produce Preharvest Environment – NYS
588 total samples collected (178 soil, 175 drag swabs, 174 water and 61 fecal samples)
44
When and where is
Listeria found?
• Temperature is the best global
predictor.
• Proximity to pasture and water are
important at permissive
temperature.
• Near average summertime
temperature and < 168 m water was
28% prevalence.
• Temperature > Average and < 440 m
from pasture had 18% prevalence.
45
Classification Tree Predictions of Reservoirs
46
Salmonella
• The genus Salmonella is divided into 2 species
• Salmonella bongori
• Salmonella enterica, which is subdivided into 6 subspecies
(enterica, salamae, arizonae, diarizonae, houtenae, indica)
• Over 2,500 recognized serotypes, e.g. Salmonella enterica subsp.
enterica serotype Typhimurium (Salmonella Typhimurium)
• Salmonellosis is one of the most common and widely distributed
foodborne diseases
• Estimated 1 million cases and 375 death annually the US
• Salmonella strains resistant to multiple antibiotics are a concern
• A number of foodborne salmonellosis outbreaks have been linked
to multidrug resistant (MDR) strains
Salmonella enterica sources
• Contamination can occur through multiple pathways including
(i) primary contamination from food producing animals (poultry,
cattle); (ii) animals that contaminate food through contact
(manure application, rodents and pests, contaminated water);
(iii) direct or indirect contamination from human sources; and
(iv) environmental sources
– Increasing recognition that Salmonella contamination from
environmental sources is a problem (“geonotic” versus
“zoonotic”?)
209 cases
28 cases
49
Salmonella enterica control
• Needs to consider likely contamination sources, which differ
between products
– Raw animal commodities: focus on control in animal populations (vaccination; testing and development of Salmonella
free animal populations; reduction strategies at slaughter)
– Dry and low water activity food products (e.g., spices, nuts,
peanut butter): validated kill steps; preventing post-harvest
cross contamination
– Produce: reducing preharvest contamination (e.g., water and
environmental testing); preventing post-harvest cross
contamination (from products and environmental sources);
reduction strategies after harvest
Salmonella enterica control – notes of
caution
• Resistance to heat treatment differs significantly depending
on environmental conditions:
– Salmonella in low water activity environments (spices, nuts, dry
powders) is significantly more resistant to heat treatment as
compared to Salmonella in high water activity environments (e.g.,
fluid milk)
• Plan for actions to be taken after an environmental positive
need to be in place before testing programs are initiated
Outline
• Introduction & overview of foodborne pathogens of
concern
• Use of DNA fingerprinting methods to detect disease
outbreaks and analyze contamination patterns
• Key pathogens
– Listeria monocytogenes
– Salmonella
• Developments in molecular detection
• Conclusions
Current challenges in detection
• Time to result
• Cost
• Expertise required for testing (e.g., interpretation of
colony morphology on plating media)
• False positives (e.g., Citrobacter that are phenotypically
very similar to Salmonella)
• False negatives (due to low bacterial numbers or due to
bacteria that behave atypically, e.g., lactose positive
Salmonella)
Molecular approaches can address many of these challenges
The target!!
The Central Dogma
DNA
Molecular
methods
DNA replication
Transcription
mRNA
Translation
Classical
methods
Protein/Enzymes
Toxins and other metabolites
Overview of Technologies
• Detection of surface molecules and other antigens
– Antibody-based methods (e.g., ELISA)
– Recombinant phage protein
• Nucleic acid hybridization methods
– Colony hybridization methods
– Dip-sticks
• Nucleic acid amplification methods
– Polymerase chain reaction (PCR)
– Other nucleic acid amplification methods
• Microarrays
– Generally lack sensitivity for detection purposes unless
combined with an initial amplification step
Isothermal Amplification Methods
• Examples include:
– Nucleic Acid Sequence-Based Amplification (NASBA)
– Transcription Mediated Amplification (TMA)
– Loop loop-mediated isothermal amplification (LAMP)
• Isothermal – does not require thermocycler
• Utilizes enzymes different from PCR:
– For example, LAMP uses Bst polymerase
3M Food Safety
How does it work?
Bioluminescence Detection
Isothermal DNA Amplification
Multiple primers recognize distinct regions of
the genome and DNA polymerase to provide
efficient, rapid and continuous amplification of
target DNA
Thermostable luciferase uses
Adenosine Tri Phosphate (ATP)
to generate light which is detected, indicating
target DNA
3. An enzyme called
ATP Sulfurylase
converts into ATP
1. Pyrophosphate
ions (PPi) generated
via DNA
amplification
58
© 2012 3M. All Rights Reserved.
2. Combine with
Adenosine 5'
phosphosulfate (APS)
4. Thermostable
luciferase uses ATP to
generate light
3M Food Safety
How does it all work?
Adenosine 5'
phosphosulfate (APS)
Specific DNA Isothermal DNA
Primer
Polymerase
Firefly Luciferase
ATP Sulfurylase
dNTP’s
Bacterial DNA Strand
At
aThe
constant
of
60°C
the
Isothermal
DNA
Polymerase
produces
complimentary
Luciferase
is
the
enzyme
allows
fireflies
to glow.
The
enzyme
consumes
high
energy
During
the
lysis
step
any
DNA
isthat
released
from
within
the
bacterial
cells
then
aabind
small
20µL
sample
dNTP’s
ATP
primer
Sulfurylase
are
is
the
the
base
component
is
an
molecules
enzyme
that
used
that
gives
is
tothe
able
the
build
assay
to
DNA
produce
its
strands.
specificity.
ATP
As
molecules
each
It
will
dNTP
only
from
base
Pyrophosphate
to
a
added
highly
toDNA
the
Adenosine
5'temperature
phosphosulfate
(APS)
is
the
precursor
molecule
that
gets
combined
with
the
PPi
to of
As
the
ATP
molecules
are
produced
they
get
consumed
by
the
Luciferase
enzyme
to
produce
The
The
ATP
Isothermal
Sulfurylase
DNA
enzyme
Polymerase
combines
produces
PPi
many
molecules
with
of
APS
the
target
molecules
DNA
in
toas
ais
produce
very
short
high
As
each
dNTP
base
is
added
to
the
new
DNA
strand
acopies
PPi
molecule
isof
released
a
by
product
Once
primer
has
bound
the
Isothermal
DNA
Polymerase
is
able
to
attach
itself.
Inthe
the
first
step
the
primer
binds
to
a
highly
specific
region
target
DNA.
strand
by
ATP
adding
molecules
dNTP
to
nucleotides
emit
photons
to
the
of
light.
sequence.
isofand
transferred
to
the
assay
tube.
strand
specific
by
ions
the
genetic
(PPi)
DNA
polymerase
sequence
adenosine
found
enzyme
5´-phosphosulfate
intemperature
the
a PPi
target
organism.
(APS)
isinstrument.
released.
ATP.
photons
of period
light.
This
emitted
light
is
detected
inmolecule
the
MDS
time
at create
a
constant
of
60°C
energy
ATP.
the
process.
59
© 2012 3M. All Rights Reserved.
Experimental design
• 391 samples were collected from retail (n=120), seafood
processing (n=72), meat processing (n=100) and dairy
processing (n=99) environments using 3M Sponge-Sticks with
D/E Neutralizing Buffer.
• Sponges were enriched in 225 mL 3M Modified Listeria
Recovery Broth directly in the 3M sample collection bag at 30
C and tested at 22 and 48 h with (i) 3M Molecular Detection
System and (ii) plating on Oxford followed by species
identification
• For 288 sample sites duplicates sponges were tested using the
60
BAM method
64
Diversity of Listeria spp. detected
• 28 L. innocua (12 allelic
types), 9 L. welshimeri (4
allelic types), 2 L. seeligeri (2
allelic types)
• 36 L. monocytogenes
representing 7 allelic types;
23 and 13 isolates,
representing lineages I and II,
respectively.
Summary
• The 3M Molecular Detection Assay Listeria, performs equally as
well as the gold standard method when used with sponge
samples collected from naturally contaminated environmental
sites.
– system allowed detection of a diversity of Listeria species
– real-time positive results were reported as early as 25 min,
following enrichment and a simple lysis protocol.
• Samples from a variety of different food associated
environments showed no assay inhibition
– Consistent with previous reports that LAMP is highly robust and
less sensitive to inhibition as compared to many PCR-based
amplification methods
Putting it all together – designing
environmental sampling plans
• Effective environmental sampling plans can prevent food
contamination before it occurs
• Sampling plans need to be developed individually for each
plant
– Layout, production schedules, facility design
• For many products Listeria and Salmonella as key targets
– Environmental sampling for spoilage organisms may also be relevant
• Trend is towards regulatory agencies recommending
environmental sampling
– Regulators may perform sampling if there are no data supporting that
sampling is done by the facility
Goals of a microbial environmental
testing program
• Identify problem areas harboring pathogen sources
and locate contamination sources
– Need to set up a system that encourages collection of
samples that yield positive results
• Confirm effectiveness of problem-solving procedures
• Secondary goal may be to characterize transmission
pathways
Where to test?
• Food contact surfaces
– Food contact surface positives may have to be followed up with
finished product testing
• Non-food contact surfaces
– Sites in coolers (floors, walls, cooler coils, condensate collectors etc.)
– Tubs, conveyances, underneath tables
– Floors, floor mats, walls, & drains in production areas
• Sites are typically pre-determined, but may be renadonly
totated so that not all siets are sampeld every times
– Fort exmapel only 15 of 30 siets may be smapled every time
Where to test – the zone concept
• Plant is divided into different zones; zones are defined based on
relative potential for finished product contamination a site or area
represents; sampling and corrections triggered by positive samples
differ by zones.
–
–
–
–
Zone 1: Finished product contact surfaces
Zone 2: Non-food contact surfaces in finished product area
Zone 3: Product contact surfaces in raw product handling areas
Zone 4: Areas remote from finished product handling (e.g., non-product
contact surfaces in the raw product handling areas)
• Some plans have 3 not 4 zones
When to test?
• Pre-op
– Less likely to yield positive samples
– More easy to interpret, will identify sanitation
weaknesses
• Mid-op
– More likely to yield positive
– Will provide information on spread of Listeria during
processing
How often to test?
• Can range from daily/multiple times a day to weekly
or maybe even monthly (in very small operations)
• Sites are typically pre-determined, but may be
randomly rotated so that not all sites are sampled
every times
– For example, only 15 of 30 predetermined sites may be
sampled every time
What to do with testing results
• Review testing results every time results are
reported
– This should include review of last 4-8 sampling results to
identify trends (e.g., site that has positives with
intervening negatives
– Take corrections on each positive sample and document
action
What to do with testing results (cont’d)
• Organize testing results in one location (folder,
three-ring binder)
– Include documentation of corrections in same location
• Conduct regular (quarterly, yearly; depends on
testing frequency & volume) review of testing results
– Tabulate and evaluate long-term trends
Guidelines for follow-up and corrections
• Corrections based on positive samples need to be
plant specific
• Each positive sample should be followed up with
additional investigations
• Trend towards increased frequency of Listeria spp.
needs to be investigated to determine reason and
action needs to be taken to reduce frequency
Guidelines for follow-up and
corrections (cont’d)
• Additional samples should be taken from environmental
area that showed positive results
• Additional positive samples after corrections need follow
up with intensified cleaning and re-testing
– Problems areas may have to be shut down temporarily
• Consider if a test and hold program is needed
Example of correction plan
Finished product testing
78
Summary and conclusions
• Pathogens of interest and pathogen sources differ between foods
– Need to know your food(s)
– Need to know public health and regulatory risks associated with
your foods
• Testing is a critical component of food safety
– Testing of food processing plant environments is critical to minimize
cross contamination
– Testing of raw commodities is important for products that do not
have effective kill steps (e.g., fresh produce)
– Need to define written procedures for positive tests before testing is
initiated
– New molecular methods can offer significant advantages