Download ProSafeBeef, Pillar 2, WP2.5, D2.5.6 AUA (Vienna, 25-03

Vienna, 25 - 26 March 2010
PILLAR 2: Control and intervention strategies
along the fork-to-farm chain to ensure beef safety
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
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 bacterial species which are typically
found in complex food industrial ecosystems
o
representatives of pathogens
o
Listeria monocytogenes (11 strains)
Salmonella enterica (8 strains)
Staphylococcus aureus (3 strains)
o
Pseudomonas sp. (6 species/strains)
representatives of spoilage bacteria
o
P. fluorescens, P. fragi, P. aeruginosa, P. phsychrophilla, P. gessardii, Pseudomonas sp.
o
Lactobacillus sakei (11 strains)
representatives of useful
technological bacteria
* All tested strains had been previously identified by 16S rRNA analysis and separated by PFGE
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
UNKNOWN
(28.2%)
11
20
3
HUMANS
(7.7%)
* Code used FMCC_B, Food Microbiology Culture
Collection_Bacteria
Laboratory of Microbiology &
Biotechnology of Foods
Agricultural University of Athens
FOODS (51.3%)
5
FOOD
INDUSTRY
SURFACE
(12.8%)
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 foodprocessing 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 (A575nm)
PS microplate with stained biofilm cells
Laboratory of Microbiology &
Biotechnology of Foods
Agricultural University of Athens
microplate reader
PILLAR 2, WP2.5, D2.5.6
Biofilm formation assay using PS microplates

Temperature: 15oC

Growth media: TSB and 1/10 dTSB

Initially, bacteria were left to adhere on PS microplates for
3 h (at 15oC). For this bacterial suspension of ca. 108cfu/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
Laboratory of Microbiology &
Biotechnology of Foods
Agricultural University of Athens
A575nm
Nutrient limited growth medium
(1/10 TSB)
Laboratory of Microbiology &
Biotechnology of Foods
Agricultural University of Athens
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-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-230
FMCC B-248
FMCC B-228
FMCC B-225
FMCC B-229
FMCC B-230
FMCC B-248
FMCC B-228
FMCC B-225
FMCC B-229
Pseudomonas sp.
FMCC B-236
FMCC B-29
FMCC B-29
Staph.
aureus
FMCC B-236
FMCC B-135
FMCC B-135
FMCC B-194
FMCC B-95
FMCC B-137
FMCC B-194
FMCC B-134
FMCC B-67
FMCC B-67
FMCC B-137
3.5
FMCC B-134
FMCC B-62
Salm. enterica
FMCC B-95
FMCC B-56
FMCC B-17
FMCC B-17
FMCC B-62
FMCC B-127
FMCC B-127
FMCC B-56
FMCC B-166
FMCC B-166
FMCC B-19
FMCC B-169
FMCC B-169
FMCC B-42
FMCC B-124
FMCC B-124
L. monocytogenes
FMCC B-42
FMCC B-164
FMCC B-164
Results: biofilm formation on PS microplates
4
FMCC B-19
FMCC B-129
FMCC B-129
0
FMCC B-165
0.5
FMCC B-130
1
FMCC B-165
1.5
FMCC B-126
2
FMCC B-130
2.5
FMCC B-125
3
FMCC B-126
3.5
FMCC B-160
4
FMCC B-125
0
FMCC B-160
A575nm
Rich growth medium (TSB)
PILLAR 2, WP2.5, D2.5.6
Lactobacillus sakei
3
2.5
2
1.5
1
0.5
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_B46) 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
Biofilm formation assay using SS coupons
SS coupons in TS broth
Laboratory of Microbiology &
Biotechnology of Foods
Agricultural University of Athens

Sterile SS coupons were fully
immersed in bacterial suspensions
of ca. 108 cfu/ml in ¼ Ringer
solution for 3 h at 15oC
(ATTACHMENT STEP)

Loosely attached cells were
removed by rinsing (with ¼ Ringer)

Coupons were then incubated in
TSB at 15oC for 6 days (144 h)
(BIOFILM FORMATION STEP)

Growth medium was renewed
every 48 h
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
Laboratory of Microbiology &
Biotechnology of Foods
Agricultural University of Athens
Agar plating
2
ΒΙΟ-ΥΜΕΝΙΟ
)
(logcfu/cm
cfu/cm
2)
BIOFILM (log
0
Laboratory of Microbiology &
Biotechnology of Foods
Agricultural University of Athens
FMCC B-29
FMCC B-34
FMCC B-26
FMCC B-55
FMCC B-29
FMCC B-34
FMCC B-26
FMCC B-55
FMCC B-229
FMCC B-225
FMCC B-228
FMCC B-248
FMCC B-230
FMCC B-236
FMCC B-239
FMCC B-227
FMCC B-238
FMCC B-43
FMCC B-229
FMCC B-225
FMCC B-228
FMCC B-248
FMCC B-230
FMCC B-236
FMCC B-239
FMCC B-227
FMCC B-238
FMCC B-237
FMCC B-226
Staph. Pseudomonas sp.
aureus
FMCC B-237
FMCC B-226
FMCC B-43
FMCC B-46
FMCC B-135
FMCC B-135
FMCC B-46
FMCC B-95
FMCC B-134
FMCC B-194
70
FMCC B-134
FMCC B-194
FMCC B-137
Salm. enterica
FMCC B-95
FMCC B-67
FMCC B-137
FMCC B-62
FMCC B-62
FMCC B-67
FMCC B-56
FMCC B-56
FMCC B-17
FMCC B-19
FMCC B-127
FMCC B-17
FMCC B-42
FMCC B-166
FMCC B-127
FMCC B-42
FMCC B-169
FMCC B-166
L. monocytogenes
FMCC B-19
FMCC B-124
FMCC B-130
FMCC B-126
FMCC B-169
1
FMCC B-164
2
FMCC B-124
3
FMCC B-129
4
FMCC B-164
5
FMCC B-165
6
FMCC B-129
7
FMCC B-165
FMCC B-130
FMCC B-125
80
FMCC B-126
8
FMCC B-160
9
FMCC B-125
0
FMCC B-160
ATTACHMENT (%)
(%)
ΠΡΟΣΚΟΛΛΗΣΗ
PILLAR 2, WP2.5, D2.5.6
Results: Attachment to and biofilm formation on SS coupons
Lactobacillus sakei
60
50
40
30
20
10
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)
PILLAR 2, WP2.5, D2.5.6
Results: Attachment to and biofilm formation on SS coupons
2
Species
Attachment (%)
Biofilm (log cfu/cm )
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
Variations at levels of attachment and biofilm formation for each species
Relationship between attachment
& biofilm forming ability for the 39
bacterial strains
BIOFILM (log cfu/cm2 )
9
8
7
6
5
4
3
20
30
40
50
60
ATTACHMENT (%)
Laboratory of Microbiology &
Biotechnology of Foods
Agricultural University of Athens
70
80
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
DUAL SPECIES
- L. monocytogenes – S. enterica
- L. monocytogenes – S. aureus
 Study multispecies
- L. monocytogenes – Pseudomonas sp.
biofilm formation on SS
- 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
Laboratory of Microbiology &
Biotechnology of Foods
Agricultural University of Athens
PILLAR 2, WP2.5, D2.5.6
Acknowledgments
BSc Student Elli Braxou
Thank you
Laboratory of Microbiology &
Biotechnology of Foods
Agricultural University of Athens