GAMIDI
Kimon Andreas Karatzas
Listeria monocytogenes
•
•
•
•
•
•
Gram positive
Facultative anaerobic
Motile
Foodborne pathogen
Causes listeriosis (0.02% of food-related diseases)
High fatality rate (28%) between immunocompromised
individuals
• 10% of food-borne-related deaths in the US
Genes under investigation
• Lmo0913: Succinate semialdehyde dehydrogenase (Acid
tolerance)
• Lmo0796: Homology to TT1927b of Thermus thermophilus HB8
possibly involved in C40 isoprenoid transport or/and storage or
metabolism (Acid tolerance)
• Lmo2391:?????(Acid tolerance)
• Lmo2748: contains motif for Pyridoxamine 5'-phosphate oxidase
(Osmotolerance)
• Started complementation of the mutants
Invasiveness of mutants
• Invasiveness of ΔsigB was
lower than the WT
0.6
0.5
• Invasiveness of all mutants
was higher than WT
% invasion
0.4
0.3
0.2
0.1
w
t
0
Strain
•
Collaboration with Dr. Aiofe Boyd and Ksenia
Matlawska-Wasowska
• Invasiveness of Δlmo0913
was the highest (5-fold
higher than the WT)
Roles of glutamate
and GABA
glutamate
γ-amino butyric acid (GABA)
GadC
glutamate
H+
Succinate semialdehyde
Lmo913
GadA
GadB
γ-amino butyric acid (GABA)
Succinate
GABAase assay
• Δlmo0913 showed lower
GABA concentrations
0.25
0.24
0.23
0.22
Od 340
0.21
wt average
0.2
913 ave
SigB ave
0.19
• Differences were not
statistically significant
0.18
0.17
0.16
0.15
0
20
40
60
80
time (min)
100
120
140
• GABAase assay should be
performed under conditions
that Δlmo0913 is induced
Future work
• Investigation of role of PLP (active vitamin B6) in
combination with Lmo2748 (Pyridoxamine 5'phosphate oxidase)
• Overexpression of all genes that were mutated
• Investigation of increased invasiveness of
Lmo0913
Experience
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•
•
•
•
Proteomics (protein 2D gel electrophoresis)
Functional genomics in Gram+ and GramTranscriptomics (RT PCR)
High Pressure technology
Antimicrobial resistance (contribution of
disinfectants to antibiotic resistance)
• Stress resistance of bacteria
Thank you
Isolation of piezotolerant strains
of L. monocytogenes
Growth in BHI until exponential
phase (30oC)
Determination of
piezotolerance
of isolates
Incubation at 37oC
Plating on BHI agar
plates
HHP treatment
350 MPa (20 min)
Isolation of individual
colonies
Listeria monocytogenes
Pure culture of
L. monocytogenes
6
Wt
4
2
AK01
0
100
200
300
400
Pressure (MPa)
Listeria monocytogenes
Log reduction
AK01: A piezotolerant L.
monocytogenes mutant
Increased thermotolerance
of AK01 at 55oC
9
7
AK01 (heat)
5
Wt
AK01
3
0
10
Tim e (m in)
20
Listeria monocytogenes
Log (cfu ml
-1
)
Wt (heat)
Phenotype of AK01
Wt
Wt
AK01
AK01
Listeria monocytogenes
Impaired growth and formation of small colonies
Reduced virulence of AK01
6
Wt
5
4
3
<
AK01
2
0
1
2
Time (Days)
3
Listeria monocytogenes
Log (cfu spleen-1)
7
Morphology of AK01
Immobility and increased cell
size
Wild type
AK01
Listeria monocytogenes
Altered protein expression
in AK01
AK01
pI gradient
Mw
pH 4
pH 6
pH4
pH6
29.0
18.4
FlaA
FlaA
ClpP
ClpP
Wt
AK01
14.3
• Decreased
FlaA
(immobility)
Listeria monocytogenes
43.0
• Increased
ClpP (stress
resistance)
A mutation in the ctsR
gene of AK01
• ctsR (class three stress gene repressor)
• MB18: insertion of the mut ctsR gene
1. Increased piezotolerance (ClpP)
2. Immobility (FlaA)
Listeria monocytogenes
• Mutation in ctsR responsible for:
Mutation in ctsR of AK01
Ser Lys Arg Gly Gly
Gly
Gly
Wt: AGT AAA CGT GGT GGT GGT GGC
Ser Lys Arg
Gly Gly
Gly
In ΑΚ01 the CtsR protein contains a glycine
less than in the Wt
Listeria monocytogenes
ΑΚ01: AGT AAA C . . . GT GGT GGT GGC
Role of ctsR
ctsR
lmo0230 lmo0231
clpC
CtsR
Repression of clpB, clpP, clpE
• Clps form complexes (essential for heat shock
response)
• ClpP (proteolytic subunit)
• ctsR is an economy mechanism by repressing
expression of heat shock proteins
Listeria monocytogenes
Autorepression
Was isolation of AK01 a rare
incident?
• HHP experiments were performed and isolates
were analyzed
Listeria monocytogenes
• Experiments performed in triplicate
• Growth temperature: 30oC
• HHP applied on pure cultures at exponential
phase
• 350 MPa for 20 min
• Randomly selected 28 Isolates each time
~30% of isolates were
piezotolerant
7
6
5
4
3
2
1
0
Wt AK
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Isolates
Listeria monocytogenes
-1
log reduction (cfu ml )
8
High frequency of mutations in ctsR
between piezotolerant isolates
Listeria monocytogenes
Increased frequency of
piezotolerant mutants
• 0.06% of cells in a Wt population are piezotolerant
(exponential phase)
• The occurrence of AK01 and other piezotolerant isolates
in a wild type population of L. monocytogenes Scott A is a
common phenomenon with stable frequency
Listeria monocytogenes
• 66% of the piezotolerant isolates contained the ctsR
mutation
The glycine-rich region of
ctsR is a Short Sequence
Repeat (SSR) region
Listeria monocytogenes
• SSRs are hypermutable
• Mechanism: DNA polymerase slippage
• Molecular studies in Saccharomyces cerevisiae have
shown that poly(G-T) tracts are extremely unstable,
with length alterations occurring at a frequency of at
least 10-4 event per cellular division (Henderson, S. T.,
and T. D. Petes. 1992).
The role of SSRs
Listeria monocytogenes
• The SSR of ctsR is a part of a strategy for the increased
production of stress tolerant mutants within populations
of L. monocytogenes
• SSRs are overepresented in stress genes (Rocha et al.
2002)
• Through SSRs, variability within a population becomes
an inherited characteristic and does not rely on the
extremely rare occurrence of an advantageous random
mutation
Conclusions
• Discovery of a piezotolerant mutant of L. monocytogenes
(AK01) with multiple stress resistance (HHP, heat, acid)
• A mutation in the ctsR gene of AK01 responsible for its
phenotype
• The mutation is in a hypermutable region resulting in
variability within the population
Listeria monocytogenes
• Immobility and reduced virulence of AK01
Does the SSR of ctsR plays a
role in stress tolerance of other
bacteria?
•
S. aureus contains 4 repeats in the
glycine rich region
higher frequency of mutagenic events?
Listeria monocytogenes
•
S. aureus
• Gram positive
• Non motile, coccus
• Natural part of human microflora
• Leading cause of nosocomial infections
• Increased antibiotic resistance (MRSA)
Staphylococcus aureus
• Foodborne pathogen: production of thermostable
toxin
In search for piezotolerant
isolates of S. aureus
• Isolation of a pure culture
• HHP of 400 MPa for 30 min
• Investigation of isolates
Staphylococcus aureus
• Growth for 5 days at 37oC (subculturing every day
following overnight incubation)
Piezotolerant variants form
small colonies
Staphylococcus aureus
2
1.5
• Small colony
variants
1
0.5
0
-0.5
-1
-1.5
1
2
3
4
5
6
7
8
9
10 11 12
13 14 15 16 17 18
19 20 21
Isolate
number
• Wild type
Staphylococcus aureus
Wt log reduction (cfu ml-1) – isolate log reduction (CFU ml-1)
Isolation of piezotolerant
variants
3
2
1
0
1 2 3 4 5 6 7 8 9 1 01 11 21 31 41 51 61 71 81 92 02 1
-1
-2
-3
-4
-5
Isolate
number
• Small colony
variants
• Wild type
Staphylococcus aureus
Wt log reduction (cfu ml-1) – isolate log reduction (CFU ml-1)
Increased thermotolerance of
piezotolerant variants
Piezotolerant S. aureus
variants are less invasive
10
8
7
6
5
4
WT
3
8
9
13
20
21
Isolate number
S. aureus can:
1. invade Caco-2 cells
2. colonise the intestinal tract, even causing enteritidis
3. be protected from antibiotic treatment while being inside the host cells
Staphylococcus aureus
Log (CFU ml-1)
9
• Invasion was
assessed in
Caco-2 cells
following the
gentamicin
protection
assay
Piezotolerant S. aureus
variants are more susceptible
to antibiotics
Amikacin
30
Kanamycin
30
Oxacillin
1
Neomycin
10
Gentamycin
10
Furazolidone
100
WT
15 R
15 I
16 R
13 R
17 R
17
1
18 I
18
21
16 R
21
17
20
15 R
18
18
13 R
17 R
17
21
20
20
21
18
21
18
Zones of inhibition were greater for the piezotolerant variants
Staphylococcus aureus
Isolate
Genomic indexing (microarrays)
was performed for isolate 21 and
the Wt
Staphylococcus aureus
• Genes SAR0666 – SAR0673 showed no hybridization
in isolate 21 in contrast to the Wt
Sequencing reveals the
mutation in isolate 21
SAR0664 SAR0665 SAR0666 SAR0667 SAR0668 SAR0669 SAR0670 SAR0671 SAR0672 SAR0673 SAR0674
ATTGCGGGTG
ATTGCGGGTG
SAR0665
SAR0675
ATTGCGGGTG
• The deletion covers 9351 bp and 10 genes
• Mutation was present in all piezotolerant isolates
Staphylococcus aureus
SAR0664
Sequencing reveals the
mutation in isolate 21
Functional annotation
SAR0665
SAR0666
SAR0667
SAR0668
SAR0669
SAR0670
SAR0671
SAR0672
SAR0673
SAR0674
(pitA)
putative esterase, similar to lipase LipA (partly deleted)
hypothetical protein
putative acetyltransferase
hypothetical protein
hypothetical protein, similar to two-component response regulator (ytsA)
putative membrane protein, putative sensor histidine kinase (ytsB)
putative ABC transporter protein (ytsC)
putative ABC transporter permease (ytsD)
conserved hypothetical protein
putative phosphate transport protein, similar to low-affinity inorganic phosphate transporter (partly deleted)
• Piezotolerance and stress tolerance is not due to mutations in ctsR
(sequencing)
• Deletion of yts operon – increased susceptibility to antibiotics of isolate 21
(ABC transporter playing role in bacitracin resistance)
Staphylococcus aureus
Gene
Gene microarrays for isolate 21
Percentage
change in gene
expression
Gene
Functional annotation
PhoB
Alkaline phosphatase III pseudogene precursor
417
SAR0110
Putative Na/Pi cotransporter protein
571
SAR0584
Hypothetical protein
862
SAR1398
Putative phosphate transporter similar to negative regulator of phosphate
(PhoU)
726
SAR1399
ABC transporter ATP-binding protein (PstB)
902
SAR1400
ABC transporter permease (PstA)
1082
SAR1401
ABC transporter permease (PstC)
1217
SAR1402
Phosphate binding lipoprotein (thioredoxine reductase) (PstS)
1194
• Genes SAR1398 – 1402 possibly comprise the Pst operon
(homology to E. coli)
• Pst operon (High-affinity inorganic phosphate transport)
• It is possibly upregulated due to loss of function of the lowaffinity Pi transporter (SAR0674, pitA)
Staphylococcus aureus
Operon
Genes upregulated in isolate 21
Gene microarrays for isolate 21
Percentage
change in gene
expression
Gene
Functional annotation
hld
delta-hemolysin precursor (virulence factor)
14
spa
immunoglobulin G binding protein A precursor (virulence factor)
10
rnaIII
rnaIII accessory gene regulator (agr) locus, delta lysin (virulence factor)
10
plc
1-phosphatidylinositol phosphodiesterase precursor (virulence factor)
30
nuc
thermonuclease precursor
21
splB
serine protease SplB
22
hysA2
hyaluronate lyase precursor 2 (virulence factor)
44
SAR0694
putative exported protein
36
SAR0304
putative exported protein
22
nrdD
anaerobic ribonucleoside-triphosphate reductase
21
narJ
respiratory nitrate reductase delta chain
35
pyrAA
putative carbamoyl-phosphate synthase, pyrimidine-specific, small chain
18
pyrAB
putative carbamoyl-phosphate synthase, pyrimidine-specific, large chain
11
dltA
D-alanine-D-alanyl carrier protein ligase
17
mprF
putative membrane protein
48
Staphylococcus aureus
agr system
Genes downregulated in isolate 21
Conclusions
• The accessory gene regulator agr (rnaIII) was downregulated
in isolate 21
Downregulation of virulence genes (hld, spa,
plc, hysA2)
Lower invasiveness
• Impossible to identify the reason for increased stress tolerance
• Piezotolerance coincides with thermotolerance (common
mechanisms of resistance)
• HHP technology
Non thermal pasteurization
Study population variability
Novel stress resistance mechanisms
Staphylococcus aureus
• Identified a potentially important region for stress tolerance,
virulence and inorganic phosphate uptake in S. aureus
S. enterica
•
•
•
•
•
Salmonella enterica
Gram negative
Motile
Facultative anaerobic
Causes Salmonellosis
Second most common cause of foodborne
disease in Europe
• 1.4 million cases in the US annually
• Increased antibiotic resistance
• 30% of isolates are antibiotic resistant (EU)
Disinfectants and antibiotic
resistance
• Lately there is growing concern that disinfectants
contribute in the emergence of antibiotic
resistance.
• Continuous growth in sublethal concentrations of
several disinfectants - Obtain mutants and study
their phenotype
Salmonella enterica
• Studies needed to evaluate this.
Disinfectants
Triclosan (Chlorophenol)
•
OXR (Potassium Peroxomonosulphate, sulphamic acid,
sodium alkyl benzene sulphonate)
•
TOP (Phenolic, tar acids, organic acids and
surfactants)
•
QACFG (Quaternary ammonium compounds, benzyl C8C18 alkyldimethyl chlorides, formaldehyde, glutaraldehyde)
Salmonella enterica
•
Disinfectant cycling experiments
• Growth of strains overnight for 7 times in
sublethal concentrations of disinfectants
• Daily subculture (100 µl into 10 ml)
• Mutant isolation on antibiotic plates (acriflavine,
tetracycline, ampicillin, kanamycin, ciprofloxacin)
Salmonella enterica
• In parallel, after 7 times subculture, subcultured
daily at raising disinfectant concentrations
MICs of populations & strains
MIC(mg/l)
MIC (%)
Pop/Single str.
Cip
Chl
Tet
Kan
Amp
EtBr
Tric
OXC
TOP
QACFG
AQAS
Wt OXC 7d
Pop
<0.03
8-16
2-4
2-4
2-8
>1024
0.25
0.20
0.40
<0.05
0.40
Wt TOP 7d
Pop
<0.03
8
2
2-4
2
>1024
0.12
0.20
0.40
<0.05
0.2-0.4
Wt QACFG 7d
Pop
<0.03
16
2-4
2-4
16
>1024
1.00
0.20
0.40
<0.05
0.40
Wt 16T 16d
Pop
<0.03
16
2-4
4
8-16
>1024
64.00
0.20
0.40
<0.05
0.2-0.4
Wt LB 7day
Pop
<0.03
8
2
2
2-4
>1024
0.25
0.20
0.40
<0.05
0.2-0.4
Wt LB 1day
Pop
<0.03
8
2
2
2-4
>1024
0.06
0.20
0.2-0.4
<0.05
0.20
OXCR1
Single str
0.06
32
2
1
8
>1024
0.25
0.20
0.40
<0.05
0.40
QACFGR2
Single str
<0.03
32
16
2
8
1024
0.25
0.20
<0.05
<0.05
0.10
TOPR2
Single str
<0.03
16
16
2
4
1024
0.25
0.20
0.10
<0.05
0.10
TRICR1
Single str
<0.03
16
2-4
4
8-16
>1024
64.00
0.20
0.40
<0.05
0.2-0.4
Colour explan.
•
X2
X4
X8
>X16
Treatment with disinfectants increased the MICs of several antibiotics
Triclosan
•
Highest potential for selection of mutants
QACFG
Salmonella enterica
Strain
Increased expression of acrAB in
isolates (RT PCR)
70000
60000
50000
40000
30000
20000
10000
R
1
Q
AC
FG
R
2
TO
P
R
2
TR
IC
R
1
TR
IC
R
2
O
XC
W
T
0
Strain
• TOPR2 had normal expression of acrAB
Salmonella enterica
Absorbance (arbitrary units)
80000
Growth impairment of isolates
o
Growth curves of isolates at 37 C
1.2
1.0
WT
QACFGR2
SKR2
TOPR2
FFSR2
OXCR1
VSR1
TRIC1
OD600
0.8
0.6
0.4
TRIC2
0.0
0
5
10
15
time (h)
• QACFGR2, TOPR2 and OXCR1 had impaired growth
• TRIC1 and TRIC2 had normal growth
Salmonella enterica
0.2
Lower motility of isolates
Wt
Wt
TOPR2
OXCR1
TRICR2
• TRICR1 and TRICR2 had normal motility
Salmonella enterica
QACFGR2
TRICR1
Colony morphology of isolates
WT
TRICR1
TOPR2
TRICR2
• TRICR1 and TRICR2 had normal size colonies
• All isolates had smooth and smaller colonies
Salmonella enterica
QACFGR2
OXCR1
Populations from QACFG and
TRIC are less invasive
7
-1
Log cfu ml invaded
6
5
4
3
2
16 TR
TRIC
IC 16
16 d
d
16
TOFF
PS 7
7d d
Q
AC S
FGK
77dd
OV
XCS
77dd
W1
td
LB
W
T
Strain
•
Gentamicin protection assay was performed at 37oC with
Caco-2 intestinal epithelial cells
Salmonella enterica
1
Isolates are less invasive
7
Log cfu ml-1 invaded
6
5
4
*
*
3
*
*
2
AT
T
TRRIC
IC R2
R2
T
TRRI
ICCR
R11
F
TOFS
PRR2
2
Q
AC
FSGK
RR22
OV
XCS
RR11
W
WT
t
1
Strain
• Gentamicin protection assay was performed at 37oC with
Caco-2 intestinal epithelial cells
Salmonella enterica
*
*
Proteomic analysis of the isolates
Protein
AcrA
AcrB
TolC
Imp
OmpC
OmpF
OmpA
AhpC
SodB
AcnA
TolB
Tsx
ElaB
SopB
NfnB
Acridine efflux pump
RND family, acridine efflux pump
outer membrane channel; specific tolerance
to colicin E1; segregation of daughter
chromosomes, role in organic solvent
tolerance
Organic solvent tolerance protein
Outer membrane protein 1b (ib;c), porin
Outer membrane protein 1a (ia;b;f), porin
putative hydrogenase, membrane
component
Glutaredoxin 3
Glutaredoxin 2
Thiol peroxidase
Alkyl hydroperoxide reductase, F52a
subunit; detoxification of hydroperoxides
Alkyl hydroperoxide reductase, C22
subunit; detoxification of hydroperoxides
Superoxide dismutase, iron
aconitate hydratase 1
Tol protein required for o uter membrane
integrity, uptake of group A colicins, and
translocation of phage DNA to cytoplasm,
may be part of multiprotein peptidoglycan
recycling complex
Nucleoside channel; receptor of phage T6
and colicin K
Putative inner membrane protein
Salmonella outer protein: homologous to
ipgD of Shigella
Dihydropteridine reductase/oxygen insensitive NAD(P)H nitroreductase
Percentage change in protein
expression
OXCR1 QACFGR2 TOPR2
135**
113**
355***
151***
158***
295***
-5
-42
-64**
9.3
-66**
-100†
-38*
0
-100†
-100†
-44**
-60**
-100†
-100†
-35*
-100†
317***
100‡
-100†
-100†
217*
100‡
-100†
100
-33
ND
100***
150**
100
-6
†
†
-100
533***
275*
-100
400**
350**
ND
-16
150*
138**
8
100***
225**
225*
187*
800*
100*
-100†
300*
340
-100†
•
All variants
showed
reduced
levels of
OMPs
• OXCR1 and
QACFGR2
had increased
levels of
AcrAB
Salmonella enterica
Antimicrobial
resistance
proteins
GrxC
GrxB
Tpx
AhpF
Functional annotation
Proteomic analysis of the isolates
Functional annotation
AtpC
Membrane -bound ATP synthase, F1 sector,
epsilon-subunit
Membrane -bound ATP synthase, F1 sector,
beta-subunit
Membrane -bound ATP synthase, F0 sector,
subunit b
Membrane -bound ATP synthas e, F1 sector,
gamma -subunit
Membrane -bound ATP synthase, F1 sector,
delta-subunit
General PTS family (Enzyme I) PEP protein phosphotransferase
DNA polymerase I, 3' -- 5' polymerase, 5' -3' and 3' -- 5' exonuclease
50S ribosomal subunit protein
30S ribosomal subunit protein S6
30S ribosomal subunit protein S22
Acetate kinase A (propionate kinase 2)
AtpD
AtpF
Metabolism
and maintenance
proteins
AtpG
AtpH
PtsI
PolA
RplY
RpsF
RpsV
AckA
•
Percentage change in protein
expression
OXCR1
QACFGR2
TOPR2
-68*
-64**
-68*
-50*
-57**
-82**
-54*
-54*
-70*
-47*
-43*
-53*
-63**
-45*
-73**
377***
322
11
250*
200
ND
550***
233**
166**
162
625**
322**
133*
250*
ND
55
33
ND
All variants had reduced levels of ATP synthase
Salmonella enterica
Protein
Proteomic analysis of the isolates
Protein
SipA
SipB
SipC
SipD
InvB
Cell invasion
proteins
InvG
InvH
PrgH
PrgK
PrgI
HilA
CheY
FlhA
FliC
Percentage change in protein
expression
OXCR1
QACFGR2
TOPR2
Cell invasion protein
Cell invasion protein
Cell invasion protein
Cell invasion protein
Surface presentation of antigens; secretory
proteins
Invasion protein; outer membrane
Invasion protein
Cell invasion protein
Cell invasion protein; lipoprotein, may link
inner and outer membranes
Cell invasion protein; cytoplasmic
Invasion genes transcription activator
-33
400*
157
100‡
15
133*
625**
257**
100‡
54
67
-100†
-14
ND
62
100
100‡
-22
-33
185**
ND
-22
13
-75*
100‡
11
-46
ND
ND
100‡
100‡
ND
ND
Chemotaxis regulator, transmits
chemoreceptor signals to flagelllar motor
components
Flagellar biosynthesis; possible export of
flagellar proteins
Flagellar biosynthesis; flagellin, filament
structural protein
-50
83
66
-25
ND
ND
-75***
100**
48**
• Increased levels of Sip proteins in OXCR1 and
QACFGR2
• Reduced levels of SipB, InvG, PrgK in TOPR2
Salmonella enterica
Motility-related
proteins
Functional annotation
Conclusions
• Sublethal concentrations of disinfectants select for strains
with reduced susceptibility to antibiotics
• Differences between disinfectants in increasing
antimicrobial resistance
• Identified possible mechanisms playing role in antimicrobial
resistance (OMPs, AcrAB, ATP synthase)
Salmonella enterica
• Majority of these strains have lower motility, altered colony
morphology and impaired virulence
Stress resistance & virulence
•
•
•
•
•
•
The majority of stress tolerant strains show defects in growth and virulence
characteristics
Bacteria under stress have impaired virulence
Disposable soma theory: There is a trade-off between the investments
required to obtain a given level of accuracy in protein synthesis and the
production of offspring (Kirkwood, 1977)
Metabolic/energy resources are limited in any one individual and that these
resources may be channelled into two main activities: preservation of the
soma (maintenance) or reproduction
Bacterial trade-off between proliferation and maintenance is a
consequence of alterations in RNA polymerase availability and
transcription factors, especially sigma factors, competing for RNA
polymerase acquisition. (Nystrom 2004)
Proliferation – Virulence
vs
Maintenance – Stress tolerance
Acknowledgements
Marjon Bennik
Frans Rombouts
Tjakko Abee
Vasilis Valdrmidis
Cormac Gahan
Colin Hill
Soula Tassou
Kostas Malidis
Tom Humphrey
Angelos Zervos
Nick Coldham
Martin Woodward
Luke Randall
Laura Piddock
Mark Webber
Alex van Belkum
Willem van Leeuven
Thank you
4
3
• Small colony
variants
2
1
0
1
3
4
6
7
Isolate
8
10
15
21
Staphylococcus aureus
Wt log reduction (cfu ml-1) – isolate log reduction (CFU ml-1)
Piezotolerant variants at 450
MPa for 15 min
The problem of
tailing in HHP
It is possible that tailing is due to
mutants occurring in populations
Listeria monocytogenes
• HHP inactivation curves show a
piezotolerant subpopulation with
unknown properties
Sublethal pressures
< 20 MPa:
• rotation of flagella and flagellin polymerization
20-50 MPa:
• cell division: long filaments, > lag period
50 MPa : stop DNA synthesis (*thymine)
58 MPa : stop protein synthesis (*leucine)
77 MPa : stop RNA synthesis (*uracil)
> 200 MPa : effective killing
Effects of HHP on growth of
E.coli
All piezotolerant isolates contain
the same mutation
2000
1800
1600
1400
1200
300
1
3
4
6
7
8
Isolate
10
15
21
Wt
Staphylococcus aureus
1000
Strains used
• Salmonella enterica serovar Typhimurium
(strain SL1344)
Resistant to ciprofloxacin, nalidixic acid (fluoroquinolones)
Mutation common in naturally occurring FQ resistant strains
Salmonella enterica
• A gyrA mutant was constructed in SL1344
background
(Asp
Gly at position 87)
Sublethal pressures
< 20 MPa:
• rotation of flagella and flagellin polymerization
20-50 MPa:
• cell division: long filaments, > lag period
50 MPa : stop DNA synthesis
58 MPa : stop protein synthesis
77 MPa : stop RNA synthesis
> 200 MPa : effective killing
Lethal pressures
200-700 MPa :
• Elimination of all vegetative cells
• Germination of spores
>700 MPa : Death of spores
Northern Analysis
clpP mRNA
Wt AK01
flaA mRNA
Wt AK01
Listeria monocytogenes
Regulation was at the transcriptional level
HHP applications in Food
Industry
Non-thermal pasteurization
Inactivation of enzymes
Change of functional properties
Retention of organoleptic properties (taste)
retention of nutritional value (vitamin C)
• Listeria monocytogenes is a serious problem in foods
that heat treatment is not possible – HHP (solution)
• Increased piezotolerance of strains and subpopulations
(E. coli) – L. monocytogenes?
Listeria monocytogenes
•
•
•
•
•
Increased acid resistance of
AK01 at pH 2.5
10
8
AK01 (pH 2.5)
6
Wt
4
AK01
0
15
30
Tim e (m in)
45
Listeria monocytogenes
Log (cfu ml
-1
)
Wt (pH 2.5)