ProSafeBeef, Pillar 2, WP2.5, D2.5.6 AUA

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

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WORKPACKAGE 2.5:

Potential risks associated with strategies


DELIVERABLE

2
.
5
.
6
:


Data

on

how

bacterial

interactions

contribute

to


(i)
biofilm

formation

ability

of

individual

strains,

and


(ii)
their

resistance

to

sanitizers

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

Biofilm formation & implications in beef
industry


In the meat industry, biofilms of both spoilage and pathogenic
bacteria may be related to serious problems of food
contamination (
lowered shelf
-
life of products, disease
transmission
)



In the majority of natural & industrial environments,
monospecies biofilms

are relatively rare Conversely,
microorganisms are associated with surfaces in
complex
multispecies communities



Bacterial interactions are believed to influence the
biofilm
forming capacity

of individual strains, as well as their
antimicrobial resistance

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Main objectives / tasks

1.
Investigate attachment to and biofilm
forming ability on model abiotic surfaces
of some food
-
relevant bacteria in
monoculture

and in
mixed
-
culture



1.
Evaluate
disinfection efficiency

of
some commercial disinfectants against
mono

&
mixed
-
culture biofilms


Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

39 bacterial strains* screened for biofilm
formation


These belonged to b
acterial species
which

are typically
found

in complex food industrial ecosystems


o
Listeria monocytogenes

(
11

strains)

o
Salmonella enterica

(
8

strains)

o
Staphylococcus aureus

(
3

strains)


o
Pseudomonas sp.

(
6

species/strains)



o
Lactobacillus sakei

(
11

strains)

representatives of pathogens

representatives of spoilage bacteria

representatives of

useful
technological bacteria

* All tested strains had been previously identified by 16S rRNA analysis and separated by PFGE


P. fluorescens, P. fragi, P. aeruginosa, P. phsychrophilla, P. gessardii, Pseudomonas sp.

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Isolation origin of strains


All strains were provided by
the
microorganisms
collection of Laboratory of
Microbiology and
Biotechnology of Foods*

(Department of Food Science
and Technology, AUA)
& had
been previously isolated
from different sources

FOODS (51.3%)
UNKNOWN
(28.2%)
HUMANS
(7.7%)
FOOD
INDUSTRY
SURFACE
(12.8%)
* Code used FMCC_B, Food Microbiology Culture
Collection_Bacteria

20

3

11

5

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Surfaces used


Two surfaces of different physicochemical properties
were used as
abiotic substrates

for biofilm
development:


1.
Polystyrene (PS)


-

96
-
well microplates




2.
Stainless steel (SS)



-

rectangular coupons of 3 x 1 x 0.1 cm, type AISI
-
304


-

material commonly used for the manufacture of food
-
processing equipment




Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Biofilm formation assay using PS microplates


Commonly applied method for easy screening biofilm formation by different
strains (many repetitions)



Stain biofilm cells with crystal violet, dissolving bound dye by
ethanol/acetone & quantification with
absorbance measurements (A
575nm
)

microplate reader

PS microplate with stained biofilm cells

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6


Temperature: 15
o
C



Growth media: TSB and 1/10 dTSB



Initially, bacteria were
left to adhere

on PS microplates
for
3 h

(at 15
o
C)
. For this bacterial suspension of
ca.

10
8
cfu/ml
in ¼ Ringer solution was used



Loosely attached cells were then removed by rinsing
(with ¼
Ringer)



Growth media were added, followed by incubation under
static conditions (except for
Pseudomonas sp.)

for 48 h



Growth media were renewed at 24 h


Biofilm formation assay using PS microplates

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Results: biofilm formation on PS microplates

0
0.5
1
1.5
2
2.5
3
3.5
4
FMCC B-160
FMCC B-125
FMCC B-126
FMCC B-130
FMCC B-165
FMCC B-129
FMCC B-164
FMCC B-124
FMCC B-169
FMCC B-166
FMCC B-127
FMCC B-17
FMCC B-19
FMCC B-42
FMCC B-56
FMCC B-62
FMCC B-67
FMCC B-137
FMCC B-194
FMCC B-95
FMCC B-134
FMCC B-135
FMCC B-29
FMCC B-34
FMCC B-26
FMCC B-55
FMCC B-46
FMCC B-43
FMCC B-226
FMCC B-237
FMCC B-238
FMCC B-227
FMCC B-239
FMCC B-236
FMCC B-230
FMCC B-248
FMCC B-228
FMCC B-225
FMCC B-229
A
575nm
0
0.5
1
1.5
2
2.5
3
3.5
4
FMCC B-160
FMCC B-125
FMCC B-126
FMCC B-130
FMCC B-165
FMCC B-129
FMCC B-164
FMCC B-124
FMCC B-169
FMCC B-166
FMCC B-127
FMCC B-17
FMCC B-19
FMCC B-42
FMCC B-56
FMCC B-62
FMCC B-67
FMCC B-137
FMCC B-194
FMCC B-95
FMCC B-134
FMCC B-135
FMCC B-29
FMCC B-34
FMCC B-26
FMCC B-55
FMCC B-46
FMCC B-43
FMCC B-226
FMCC B-237
FMCC B-238
FMCC B-227
FMCC B-239
FMCC B-236
FMCC B-230
FMCC B-248
FMCC B-228
FMCC B-225
FMCC B-229
A
575nm
L. monocytogenes
Salm. enterica
Staph.
aureus
Pseudomonas sp.
Lactobacillus sakei
Rich growth medium
(
TSB
)
Nutrient limited growth medium
(1/10
TSB
)
Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Results: biofilm formation on PS microplates


For most

L. monocytogenes

strains no significant differences were
observed on biofilm formation between the
2
growth media




All
S. enterica
strains (
except

FMCC_B
-
62)
formed more biofilm

(
p
<
0.05)
when cultured in

1/10 TSB
compared to

TSB




The

3
Staph. aureus
and the 11
L. sakei

strains were poor biofilm
producers in both nutritional conditions




Pseudomonas fluorescens

(FMCC_B
-
29),
Pseudomonas
aeruginosa

(FMCC_B
-
26)
and
Pseudomonas gessardii

(FMCC_B
-
46)
formed high amount of biofilm in both growth media
.
On the
contrary
,
the other

3
Pseudomonas

species produced low biofilm



Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6


Sterile SS coupons were fully
immersed in bacterial suspensions
of
ca.

10
8

cfu/ml in ¼ Ringer
solution
for 3 h at 15
o
C

(
ATTACHMENT STEP
)



Loosely attached cells were
removed by rinsing
(with ¼ Ringer)



Coupons were then incubated
in
TSB at 15
o
C for 6 days

(144 h)


(
BIOFILM FORMATION STEP
)



Growth medium was renewed
every 48 h

Biofilm formation assay using SS coupons

SS coupons in TS broth

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Quantification of biofilm formation on SS coupons


Method based on detaching attached biofilm cells by
“bead
vortexing”

followed by quantification by
“agar plating”

SS coupon in inoculated growth medium (TSB)
Removal of coupon
using forceps
Rinsing with
¼
Ringer
Vortexing (2

) with
glass beads
Agar plating
Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Results: Attachment to and biofilm formation on SS coupons

0
10
20
30
40
50
60
70
80
FMCC B-160
FMCC B-125
FMCC B-126
FMCC B-130
FMCC B-165
FMCC B-129
FMCC B-164
FMCC B-124
FMCC B-169
FMCC B-166
FMCC B-127
FMCC B-17
FMCC B-19
FMCC B-42
FMCC B-56
FMCC B-62
FMCC B-67
FMCC B-137
FMCC B-194
FMCC B-95
FMCC B-134
FMCC B-135
FMCC B-29
FMCC B-34
FMCC B-26
FMCC B-55
FMCC B-46
FMCC B-43
FMCC B-226
FMCC B-237
FMCC B-238
FMCC B-227
FMCC B-239
FMCC B-236
FMCC B-230
FMCC B-248
FMCC B-228
FMCC B-225
FMCC B-229
ΠΡΟΣΚΟΛΛΗΣΗ (%)
0
1
2
3
4
5
6
7
8
9
FMCC B-160
FMCC B-125
FMCC B-126
FMCC B-130
FMCC B-165
FMCC B-129
FMCC B-164
FMCC B-124
FMCC B-169
FMCC B-166
FMCC B-127
FMCC B-17
FMCC B-19
FMCC B-42
FMCC B-56
FMCC B-62
FMCC B-67
FMCC B-137
FMCC B-194
FMCC B-95
FMCC B-134
FMCC B-135
FMCC B-29
FMCC B-34
FMCC B-26
FMCC B-55
FMCC B-46
FMCC B-43
FMCC B-226
FMCC B-237
FMCC B-238
FMCC B-227
FMCC B-239
FMCC B-236
FMCC B-230
FMCC B-248
FMCC B-228
FMCC B-225
FMCC B-229
ΒΙΟ-ΥΜΕΝΙΟ (log cfu/cm
2
)
L. monocytogenes
Salm. enterica
Staph.
aureus
Pseudomonas sp.
Lactobacillus sakei
BIOFILM (log cfu/cm
2
)
ATTACHMENT (%)
The attachment
ability of each strain
was expressed as the
percentage
(%)
of
cells being attached
,
compared to the total
population of cells
contained in bacterial
suspension in which
the SS coupon was
immersed

(
for

3
h
)

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Results: Attachment to and biofilm formation on SS coupons

Species
Attachment (%)
Biofilm (log cfu/cm
2
)
Listeria monocytogenes
47.1 - 63.5
4.71 - 6.27
Salmonella enterica
53.6 - 64.1
4.63 - 5.64
Staphylococcus aureus
69 - 71.6
4.71 - 5.42
Pseudomonas sp.
30.5 - 74.4
4.33 - 7.81
Lactobacillus sakei
26.8 - 61.9
3.59 - 5.65
3
4
5
6
7
8
9
20
30
40
50
60
70
80
ATTACHMENT (%)
BIOFILM (log cfu/cm
2
)
Variations

at

levels

of

attachment

and

biofilm

formation

for

each

species

Relationship between attachment
&
biofilm forming ability for the
39
bacterial strains

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Future work (…to be done the next 6 months)


Select
3 strains from each species

& test biofilm formation
on SS in
monospecies mixed culture





Study
multispecies
biofilm formation

on SS

-

L. monocytogenes


S. enterica

-

L. monocytogenes


S. aureus

-

L. monocytogenes


Pseudomonas sp.

-

L. monocytogenes


L. sakei

-

L. monocytogenes


S. enterica
-

S.
aureus


Pseudomonas sp.


L. sakei


Test
disinfection efficiency
of 3 commercial disinfectants
(
benzalkonium

chloride, chlorine, PAA)
against mono
-

and
mixed
-
culture
biofilms



DUAL SPECIES

Laboratory of Microbiology &
Biotechnology of Foods

Agricultural University of Athens

PILLAR 2, WP2.5, D2.5.6

Thank you

Acknowledgments

BSc Student Elli Braxou