ProSafeBeef, Pillar 2, WP2.5, D2.5.1 AUA

noisymaniacalBiotechnology

Feb 20, 2013 (4 years and 6 months ago)

108 views

WORKPACKAGE 2.5:

Potential risks associated with strategies


DELIVERABLE

2
.
5
.
1
:



Data

on

the

efficiency

of

current

and

new

preservation/safety

interventions

against

stress

‘hardened’

or

adapted

pathogens


PILLAR 2:

Control and intervention strategies
along the fork
-
to
-
farm chain to ensure beef safety

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

Vienna, 25
-

26 March 2010

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

2

Aim of the study:


To

investigate

the

potential

effect

of

adaptive

stationary

phase

ATR

of

L
.

monocytogenes

cells

on
:




(I)

their

attachment

to

SS

under

common

food
-
relevant

stresses

(refrigeration,

low

pH,

high

salinity)



&



(II)

the

subsequent

resistance

of

sessile

cells

to

disinfection

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

3

Acid tolerance response (ATR)


One

of

the

most

important

and

intensively

investigated

adaptive

responses

(Hill

et

al
.

1995
)
.




Results

from

pre
-
exposure

of

bacterial

cells

to

mild
-
acid

conditions

(pH

5
.
0

to

6
.
0
)





Salmonella enterica

(Foster and Hall 1990)



Escherichia coli

O157:H7

(Leyer
et al
. 1995; Buchanan
et al
.
1999)




L. monocytogenes

(Gahan
et al
. 1996; O’Driscoll
et al.

1996; Lou
and Yousef 1996; 1997; Koutsoumanis
et al.

2003; Koutsoumanis and
Sofos 2004; Skandamis et al. 2008; 2009)
.

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

4

L. monocytogenes

& ATR



Besides

enabling

enhanced

resistance

to

lethal

acid

exposure

(pH



3
)

(Koutsoumanis

et

al
.

2003
;

Koutsoumanis

and

Sofos

2004
)
;

it

has

also

been

found

to

offer

cross
-
protection

against
:



heat



ethanol



oxidative and osmotic stresses



bacteriocin nisin and other ionophores

(O’Driscoll

et

al
.

1996
;

Datta

and

Benjamin

1997
;

Lou

and

Yousef

1997
;

Koutsoumanis

et

al
.

2003
;

Skandamis

et

al
.

2008
;

2009
)


D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

5



Bacterial strain:



Strain
.

Listeria

monocytogenes

Scott

A

(serotype

4
b,

epidemic

strain,

human

isolate)
.



TSB
-
G
does not result in the formation of an acidic environment
(lack of fermentable carbohydrate).




TSB+G is acidogenic, supporting growth of the microorganism with
an accompanying decrease of its pH.




Assay for ATR induction:



After a preculture in TSB, bacteria were subcultured in
either TSB+G or TSB
-
G
(Buchanan and Edelson 1996)
.

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

6

Abiotic surface & bacterial attachment


Stainless

steel

(SS)

coupons

(
3

x

0
.
8

x

0
.
1

cm,

type

AISI
-
304
)
.




Setup
:

coupons

were

placed

vertically

in

test

tubes

containing

BHI

broth



Inoculation:

ca.

10
8

CFU/ml of either
nonadapted

or
acid
-
adapted

cells.




Incubation:

statically for 10 d.

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

7

Cases examined for bacterial attachment

Case
Temperature (
o
C)
pH
Salt content (% w/v NaCl)
Attachment conditions
A
refrigeration
B
neutral
C
mild acidity
D
mild salinity
E
mild acidity & salinity

5

7.4

0.5


16

7.4

0.5


16

4.5

0.5


16

7.4

5.5


16

4.5

5.5

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

8

Exposure of attached cells to disinfection

SS coupon in BHI
for 10 d

SS coupon carrying
attached bacteria

REMOVAL

RINSING

DISINFECTION (6’)

Attached
L. monocytogenes

cells to SS
following acridine orange staining &
epifluorescence microscopy observation

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

9

Exposure of attached cells to disinfection

DISINFECTION (6’)



HCL

(pH

2
)



Lactic

acid

(pH

2
)



Essential

oil

of

Satureja

thymbra

(
1
%

v/v)



Hydrosol

of

S
.

thymbra

(
100
%
)

Satureja thymbra


Essential oils & extracts of plants, herbs, and spices
constitute a class of very potent natural antimicrobials

(Skandamis and Nychas 2001; Nychas
et al.

2003;
Burt 2004)
.



Essential oil and hydrosol of
S. thymbra

have
recently been found to present sufficient bactericidal
effect against monoculture and mixed
-
culture biofilms
(Chorianopoulos
et al
. 2008)
.


D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

10

DISINFECTION (6’)

Quantification of viable attached bacterial population

RINSING

Bead vortexing & agar plating

Conductance measurements

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

11

Bead vortexing & agar plating

Conductance measurements

Vortexing (2’)

Plate counting on TSA

Incubation into Malthus
module
(30
o
C for 48 h)


SS coupon


electrodes

Conductance
measuring tube



Detection

of

attached

cells

by

impedance

measurements

is

already

cited

in

the

literature

(Johnston

and

Jones

1995
;

Flint

et

al
.

1997
;

Giaouris

et

al
.

2005
)
.

D2.5.1

Quantification of viable attached bacterial population

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

12

Conductance measurements (MALTHUS)

HOURS

Detection time (DT)

0

5

10

15

20

1000

1500

2000

2500

3000

3500

4000

CONDUCTIVITY

(
μ
S)

PC recording
conductance changes



Corresponding data [conductance (
μ
S)
vs

time (h)] are
recorded graphically as conductance growth curves.


During

bacterial

growth,

uncharged

or

weakly

charged

substrates

(proteins,

carbohydrates

etc
.
)

are

converted

into

highly

charged

and

products

(amino

acids

etc
.
)

Increase of
broth
conductance


(Silley and Forsythe 1996)

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

13

inversely related to
the initial
bacterial

population into the
Malthus tubes



Longer

DT

suggests

less

remaining

viable

bacteria

after

disinfection

(reduced

resistance)

D2.5.1

Conductance measurements (MALTHUS)

HOURS

Detection time (DT)

0

5

10

15

20

1000

1500

2000

2500

3000

3500

4000

CONDUCTIVITY

(
μ
S)

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

14

Results

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

15

Attachment of nonadapted ( ) & acid
-
adapted ( )
L. monocytogenes

cells to SS

A
B
C
D
E
0
1
2
3
4
5
LOG (CFU/CM
2
)
CASES EXAMINED FOR BACTERIAL
ATTACHMENT
Case
Attachment conditions
A
refrigeration
B
neutral
C
mild acidity
D
mild salinity
E
mild acidity & salinity


ATR

slightly

increased

attachment

under

mil
d

acidic

conditions,

regardless

of

NaCl

concentration
.










No effect when under
natural pH conditions.

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

16

ATR & resistance of sessile cells to
disinfection



The

effect

of

each

disinfection

treatment

was

expressed

as

the

percentage

of

attached

cells

killed

through

the

action

of

sanitizer
:

SANITATION
EFFICIENCY (%)
(1
-
LOG
remaining
/LOG
initial
) x 100
=
SANITATION
EFFICIENCY (%)
(1
-
LOG
remaining
/LOG
initial
) x 100
=
D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

17

HCl
Lactic acid
Essential oil
Hydrosol
54.16
58.67
75.53*
75.53*
27.53
31.53
75.76*
75.76*
0
10
20
30
40
50
60
70
80
HCl
Lactic acid
Essential oil
Hydrosol
49.78
51.31
77.51*
77.51*
23.15
28.76
76.85*
76.85*
0
10
20
30
40
50
60
70
80
HCl
Lactic acid
Essential oil
Hydrosol
67.71*
67.71*
67.71*
67.71*
30.49
36.43
73.39*
73.39*
0
10
20
30
40
50
60
70
80
Sanitation efficiency (%)

Sanitation efficiency (%)

Sanitation efficiency (%)

: nonadapted

: acid
-
adapted

5
o
C, pH 7.4, 0.5% NaCl

16
o
C, pH 7.4, 0.5% NaCl

16
o
C, pH 4.5, 0.5% NaCl

*
Counts

after

disinfection

below

plate

counting

detection

limit

(
1
.
03

log

cfu/cm
2
)
.

A

B

C

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

18

: nonadapted

: acid
-
adapted

HCl
Lactic acid
Essential oil
Hydrosol
60.56
60.56
73.79*
73.79*
28.53
34.82
73.04*
73.04*
0
10
20
30
40
50
60
70
80
HCl
Lactic acid
Essential oil
Hydrosol
66.23*
66.23*
66.23*
66.23*
29.71
36.87
72.68*
72.68*
0
10
20
30
40
50
60
70
80
Sanitation efficiency (%)

Sanitation efficiency (%)

16
o
C, pH 4.5, 5.5% NaCl

16
o
C, pH 7.4, 5.5% NaCl

D

E

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

19



For

all

studied

cases,

cells

that

had

initially

been

acid

adapted

were

more

resistant

to

extreme

acid

challenge

(pH

2
),

compared

to

nonadapted

ones
.





Essential

oil

&

hydrosol

exhibited

strong

antimicrobial

action,

regardless

of

induction

of

ATR

response
.




The

mild

acidic

attachment

conditions

(pH

4
.
5
)

do

not

seem

to

confer

any

pH
-
inducible

acid

tolerance

to

L
.

monocytogenes

cells

that

had

not

initially

been

acid

adapted
.




Cells

that

had

initially

been

acid

adapted

conserve

their

acid

tolerance

(even

after

10

d

under

various

environmental

conditions)
.




A
cquired

acid

tolerance

of

L
.

monocytogenes

was

conserved

even

after

several

weeks

of

storage

of

the

adapted

bacteria

at

4
o
C

(Phan
-
Thanh

and

Montagne

1998
)
.




In

E
.

coli

the

acid

resistance

systems,

once

activated,

remain

active

over

prolonged

periods

of

cold

storage

(Lin

et

al
.

1996
)
.


Conclusions regarding ATR & disinfection

D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

20

Bead vortexing & agar plating
vs

conductance method

10
15
20
25
30
35
40
45
50
20
30
40
50
60
70
80
90
SANITATION EFFICIENCY (%)
DETECTION TIME (h)
48
End of test


Conductance

DTs

corresponded

well

to

agar

plating

results,

since

longer

DTs

correlated

enough

properly

with

higher

sanitation

efficiencies
.


D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Perspectives…


Use a composite of strains to simulate better the
conditions encountered in real complex food processing
ecosystems.
Different strains may respond differently
to stresses and is likely that research investigating
stress responses of only one strain may not always
provide accurately evaluation of the worst
-
case scenario.



Find and understand the molecular events probably
jointly involved in
L. monocytogenes

stress adaptive
responses

and
attachment to abiotic surfaces
.


D2.5.1

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Thank you

Acknowledgments

Dr Nikos Chorianopoulos

Dr Panos Skandamis

D2.5.1