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