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Listeria monocytogenes: Biofilmformation and persistence in theformation and persistence in the food processing environment
Mansel Griffiths
University of GuelphUniversity of Guelph
Steps in biofilm formationSteps in biofilm formation
2. Adhesion of cells2. Adhesion of cellsa) Reversible
i. Van der Waals forces
ii. Hydrophobicity
iii. Electrostatic forces
Steps in biofilm formationSteps in biofilm formation
2. Adhesion of cells2. Adhesion of cellsb) Irreversible
i. Adhesive substancesOMPs; LPS; Fimbriae; Pili; Flagella
ii. Forces involvedDipole‐dipole
Hydrogen
Ionic
Covalent
Hydrophobic
Steps in biofilm formationSteps in biofilm formation
3. Formation of microcolony3. Formation of microcolonya) Cells grow and divide
b) Layer covering surface
Steps in biofilm formationSteps in biofilm formation
3. Formation of biofilm structure3. Formation of biofilm structurea) Formation of EPS
i. Polysaccharides
ii. Proteins
iii. 98‐99% water
b) Anchors cellsb) Anchors cells
c) Protects against environmental stress
Steps in biofilm formationSteps in biofilm formation
4. Formation of mixed biofilm4. Formation of mixed biofilma) Change in mass transfer
b) Anoxic
c) Development of other speciesi. Protozoa
ii B t iii. Bacteria
iii. Fungi
Multiple phenotypes during biofilm f iformation
Reversible
Increase in protein expression
attachment
Irreversible Increase in protein expressionof 35% at each stage
Irreversibleattachment
Maturation‐1
Maturation‐2
DispersionDispersion
Cells are shed from biofilms intermittentlyCells are shed from biofilms intermittently
Listeria monocytogenes biofilms
Consequences of biofilms for the food i dindustry
• Fouling of pasteurizersFouling of pasteurizers
• Filling lines
i i• Post‐process contamination
• Fouling of packaging material
What types cause disease?What types cause disease?
• 12 serotypes (i.e. 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, yp ( / , / , / , , , , , ,4c, 4d, 4e and 7) have been recognized in L. monocytogenes
/ / b d b bl f• Serotypes 1/2a, 1/2b and 4b are responsible for 98% of documented human listeriosis cases
• Serotypes 4a and 4c are rarely associated with• Serotypes 4a and 4c are rarely associated with outbreaks of the disease
• serotype 4b strains are isolated mostly from yp yepidemic outbreaks of listeriosis, serotypes 1/2a and 1/2b are linked to sporadic L. monocytogenesinfectioninfection
Biofilm formation by L. monocytogenes
% Biofilm formation (relative to ScottA)
Lineage I Lineage II Clinical Food
Biofilm formation by L. monocytogenes
Lineage I Lineage II Clinical Food
76.3 59.2 71.9 63.0
lineage I isolates represent serotypes 1/2b 3b 3c and 4b
From Takahashi et al. 2009
lineage I isolates represent serotypes 1/2b, 3b, 3c and 4blineage II isolates represent serotypes 1/2a, 1/2c and 3a
Stainless steel
From Di Bonaventura et al. 2008
Adhesion of L. monocytogenes to surfacesAdhesion of L. monocytogenes to surfaces
From Tresse et al. 2007
Biofilm formation by L. monocytogenesBiofilm formation by L. monocytogenes
From Takahashi et al. 2009
Biofilm formation by L. monocytogenes of diff i idifferent origin
(A) environmental(B) animal(B) animal (C) raw milk (D) non-dairy food(E) clinical
From Harvey et al. 2007
Strain competition in L. monocytogenes bi filbiofilm
Biofilm production of strains of L. monocytogenes as determined by microplate assay.
Planktonic cell density of L. monocytogenes serotype 1/2a and 4b strains shed from y p y ypmixed strain biofilms.
From Pan et al. 2009
Biofilm formation by L. monocytogenesBiofilm formation by L. monocytogenes
12°C4°C
37°C22°C
From Di Bonaventura et al. 2008
Biofilm formation by L. monocytogenesBiofilm formation by L. monocytogenes
4°C 25°C
37°CBatch mode
Fed-Batch modemode
From Rodrigues et al. 2009
Biofilm formation by L. monocytogenesBiofilm formation by L. monocytogenes
Nutrient rich Nutrient depleted
Serotype 4b Serotype 1/2a
From Folsom et al. 2006
L. monocytogenes biofilm formation and dh i i l ladhesion on stainless steel
AdhesionBiofilm formation
Adhesion
Mechanicalfinished
Mechanical finished electropolished
From Rodriguez et al. 2008
finished electropolished
Transfer of L. monocytogenes to food from i l lstainless steel
From Rodriguez and McLandsborough 2007
Transfer of L. monocytogenes to food from diff fdifferent surfaces
Stainless steel
HDPE
From Rodriguez and McLandsborough 2007
Effect of injury and ability to form biofilm on the ability to t f L t d i li i f RTE ttransfer L. monocytogenes during slicing of RTE meats
From Keskinen et al. 2008
Genetic basis for biofilm formation byL. monocytogenes
From Møretrø and Langsrud 2004
Biofilm formation and quorum sensing in L. monocytogenes
From Riedel et al. 2009
Novel control strategiesNovel control strategies
• Interference with signalingg g– Furanone– Enzymes
lh l• Acylhomoserine lactonases
• Physical methods– Super‐high magnetic fieldsSuper high magnetic fields– Ultrasound– High‐pulsed electric fields – Antimicrobial surfaces
Resistance to antimicrobial agentsResistance to antimicrobial agents
• NeutralizationNeutralization
• Inability to penetrate
i li i d h i l• Nutrient‐limited physiology
• Adaptation
Resistance of Listeria monocytogenes in biofilms QUATto QUAT
Sanitizer efficacy against L. monocytogenes bi fil diff fbiofilm on different surfaces
Smooth surface
R h fRough surface
From Yang et al. 2009
Adaptation of Listeria monocytogenes to i isanitizers
5
6μg
/ml)
3
4
(ug/
ml)
um c
hlor
ide
(μ
1
2
or B
enza
lkon
iu
H7764 H7962 LJH 389 LJH 381 Lm 222 C 511
L. monocytogenes strains
0
O i i l 2 b lt BC 5 b lt BC
MIC
fo
Original 2 sub-cultures + BC 5 sub-cultures + BC
Mechanisms of sanitizer resistanceMechanisms of sanitizer resistance
Membrane structure changesEfflux pump induction
Mechanism of sanitizer resistanceMechanism of sanitizer resistance
F R t l 2006
S = sensitive; R = resistant; A = adapted; C = cured
From Romanova et al. 2006
Ribotypes of QUAT resistant and sensitive Listeria t t imonocytogenes strains
1S
Cosine coefficient
100
98969492908886848280 1.00
1.50
2.00
3.00
4.00
5.00
6.00
7.00
8.00
10.0
0
15.0
0
30.0
0
60.0
0
Molecular Size (Kb)
H
Cosine coefficient
100
98969492908886848280 1.00
1.50
2.00
3.00
4.00
5.00
6.00
7.00
8.00
10.0
0
15.0
0
30.0
0
60.0
0
Molecular Size (Kb)
H
1R
1R3M;2S
F
A
D
F
A
D
1S
1M
1R
I
L
E
K
I
L
E
KK 1S
1M
2M;1S
2R;1M
K
C
B
G
K
C
B
G
K
C
B
G ;
1S
R = MIC > 5ug/ml; M = MIC 0.8 – 4.9ug/ml; S = MIC < 0.78ug/ml
JJJ
Cell properties affect adhesionCell properties affect adhesion
Hydrophilic cells/H d hili fHydrophilic surface
Hydrophobic cells/Hydrophilic surface
Photograph by H.C. Van der Meir, R. Box, and H.J Busscher.
Persistence of L. monocytogenesPersistence of L. monocytogenes
From Møretrø and Langsrud 2004
Listeria monocytogenes distribution in poultry i lprocessing plants
Automatic evisceration Manual evisceration
From Chiarini et al. 2009
Persistence of L. monocytogenes in chilled food plants
From Keto-Timonen et al. 2007
Sources of persisters in food processing plants
() PFGE TypePersisterPersisterNon-persister
From Lundén et al. 2003
Contamination of product by persisters
From Lundén et al. 2003
Mechanism of persistenceMechanism of persistence
From Lundén et al. 2008
From Lundén et al. 2002
Mechanism of persistenceMechanism of persistence
Persister
Non-persister
From Borucki et al. 2003
serotypes 1/2b and 4b serotypes 1/2a and 1/2cSerotypes 1/2b and 4b Serotypes 1/2a and 1/2c
Mechanism of persistenceMechanism of persistence• Other factors:
– desiccation resistance
– type E monocin
– plasmid carriage
– Cd resistance
– growth and survival at low temperatures
– nutrient scavenging g g
– strains that persist in food processing plants are associated with virulence‐ attenuating mutations in inlA, required for cell invasion
PersistencePersistence
• Each plant has unique L monocytogenesEach plant has unique L. monocytogenes ecology
• A given plant tends to be colonized by a single• A given plant tends to be colonized by a single or few persistent strains
A f ifi l l b• A few specific molecular subtypes appear to persist in the general environment of food
i l (L i l 2004 Thi hprocessing plants (Lappi et al., 2004; Thimothe et al., 2004)
SolutionSolution
• L monocytogenes is ubiquitous and thusL. monocytogenes is ubiquitous and thus difficult, though not impossible, to control in food processing plant environmentfood processing plant environment
• In‐plant training sessions can improve knowledge about L monocytogenes ecologyknowledge about L. monocytogenes ecology and may lead to better control
C bi d i d l l b i• Combined testing and molecular subtyping can elucidate transmission patterns, identify h b i d i iharborage sites and recognize persisters
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