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Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids
John P. Bowman
TIAR/School of Agricultural ScienceUniversity of TasmaniaHobart, TasmaniaAustralia
Are more “hardier/problematic” strains of L. monocytogenes emerging?
Vast majority inactivatedbut maybe some surviveselect resistant /robust strains?
Source e.g.an animal
Processingstorage
Survives in food product e.g. RTE
Environmental dispersal/re-dispersal
Can “persistence” be defined andattributed to biological mechanisms
L. monocytogenes “persistent” strains• Biofilms (surface attachment) ?• Differential responses to stress?• Broader capacity to survive in food
suppy chain environmental niches?
L. monocytogenes biofilms and persistence
pH 5.0
pH 7.3
pH 8.5
stainless steel surface – different pH (24 h period)
Nilsson et al Int. J. Food Microbiol. submitted
Persistent strain
Sporadic strain
Survival in raw milk cheese (pH 4.5, 15C, aw 0.93) varies between strains
0
1
2
3
4
5
6
7
8
9
10
0 100 200 300 400 500 600
Time (h)
Via
ble
nu
mb
ers
(lo
g10
CF
U.m
l-1)
Esta Hages (PhD Thesis, University of Tasmania)
Strain (serotype):
+ FW03/0035 (4b)○ ScottA (4b)□ LO28 (1/2a)×ATCC 19115 (4b)∆ 70-1700 (4e)
terpenoidbackbones
Branched chainfatty acids
acetate
acetylphosphate
acetyl-CoApyruvatelactate
malonyl-CoA
acetoacetyl-CoAformate
oxaloacetate
TCA Cycle
2-acetolactate
acetoinBCAA
sugars
fatty acids
acs
ptb-buk-lpd-bkdA1A2-bkdB
alsD
alsS pta
lmo1381
pflA pflBC
ldh
pycA
pdhABCD
lmo1414
accABCD
ackA
lmo0722
ilvABCDHleuABCD
acetaldehyde
lmo1634
ethanol
End-products of L. monocytogenes metabolism of sugars – a common feature netween its biology and food preservation
AerobicAnaerobic
- Organic acid resistance variation between strains ….correlations?
- How does aciduric capacity affect growth patterns?
- What is the association with tolerance to non-growth permissive mineral acid (stomach acid)?
- Physiological and genetic nature of strains with variant aciduric capacities?
Organic acids and L. monocytogenes – doesaciduricity relate to strain “success” (persistence)
Source:
Genetic lineage/serotype clinical food
factory environment cattle poultry sheep
other animals(goats, dogs,
horses, wallabies)
Lineage I: No. of strains:
1/2b 1 2 1 1
3b 1
4b 5 1 9 2 11 3
4e 2 1 2 1
Lineage II:
1/2a 1 9 3 3 7 13 5
1/2c 4 1 3
3a 1 1
Lineage III:
4a 2 1 13 1
4c 3 1
Strains examined for organic acid resistance – isolated from throughout the “food supply chain”
Distribution of strain used to screen for relative organic acid resistance
Screened growth levels for 4 popular organic acidfood additives
Sodium lactate (8.9-156 mM)
Sodium diacetate (0.35-70.3 mM)
Potassium sorbate (3.3-33 mM)
Potassium benzoate (2.3-23 mM)
Efect of isolation source on acetate and sorbateresistance
Bowman et al. Appl. Environ Microbiol submitted
Abs
olu
te n
et a
bsor
banc
e
21 mM sodium diacetate
Abs
olu
te n
et a
bsor
banc
epoultry sheep cattle factory food clinical
23 mM potassiumsorbate
poultry sheep cattle factory food clinical
Ab
solu
te n
et a
bso
rban
ce
21 mM sodium diacetate
23 mM potassiumsorbate
I
II
III
I
II III
Efect of genetic lineage of isolate on acetate and sorbateresistance
0.0
0.5
1.0
1.5
2.0
5 5.2 5.4 5.6 5.8 6
gene
rati
ons h
-1
final pH
0.0
0.5
1.0
1.5
2.0
2.5
4.5 5 5.5 6
gene
rati
ons h
-1
final pH
0.0
0.2
0.4
0.6
0.8
1.0
1.2
4.5 5 5.5 6
biom
ass (
A 600
nm)
final pH
0.0
0.5
1.0
1.5
2.0
4.8 5 5.2 5.4 5.6 5.8 6
gene
rati
ons h
-1
final pH
0.0
0.2
0.4
0.6
0.8
1.0
4.8 5 5.2 5.4 5.6 5.8 6
biom
ass (
A60
0 nm
)
final pH
0.0
0.2
0.4
0.6
0.8
1.0
1.2
5 5.2 5.4 5.6 5.8 6
biom
ass (
A 600
nm)
final pH
+10 mM sodium diacetate
+20 mM sodium diacetate
+0 mM sodium diacetate
Bowman et al. Appl. Environ Microbiol submitted
Growth rate is faster in resistant strain FW04/0025 compared to EGD but growth yield is the same when stressed with sodium diacetate
Exponential phase/pH 5.0 (HCl) Stationary phase/pH 5.0 (HCl)
FW04/0025 FW04/0025
EGDEGD
The acid tolerance (pH 2.4, 2 hour exposure)of FW04/0025 was greater than EGDunder all conditions except exponential phase at pH 7.3
Exponential phase/pH 5.0 (HCl)+ 21 mM sodium diacetate
Stationary phase/pH 5.0 (HCl)+ 21 mM sodium diacetate
FW04/0025
EGD
FW04/0025
EGD
Sodium diacetate promotes acid tolerance arising fromboth pH-dependent and phase-dependent adaptation
0
50
100
150
200
250
300
350
400
450
500
EGD ATCC 19111 FW04/0023 FW04/0025
intr
acel
lula
r ac
etat
e (m
M)
0
50
100
150
200
250
300
350
400
450
500
EGD ATCC 19111 FW04/0023 FW04/0025
K+(m
M)
Resistant strains (FW04/0025, FW04/0023) accumulate less sodium diacetate and K+ compared to less resistant strains
BHI pH 7.3
BHI pH 5.0
BHI pH 5.5+21 mM SDBHI pH 5.0+21 mM SD
Sodium diacetate
K+
Microarray comparisons reveal several differences when strains are cultured in the presence of 21 mM sodium diacetate at pH 5.0
FW04/0025(pH 5.0 + 21 mM SDA
vs. pH 5.0)
EGD(pH 5.0 + 21 mM SDA
vs. pH 5.0)
FW04/0025(pH 5.0 vs. pH 7.3)
EGD(pH 5.0 vs. pH 7.3)
54 72 108
45
26
11612
6
6 2 4
5
17 22
18
Bowman et al. Appl. Environ Microbiol submitted
Number of genes differentially expressed:
EGD responds strongly to HCl acidic stress (pH 5.0) compared to FW04/0025
Responses of known pH homeostasis mechanisms in relation to different forms of acid stress:pH 5.0 – white barspH 5.0 + 21 mM sodium diacetate – black bars
- 4 - 3 - 2 - 1 0 1 2 3 4
atpC
atpD
atpG
atpA
atpH
atpF
atpE
atpB
atpI arcA
arcB
arcC
gadA
gadB
gadC
alsD
alsS
Log Ratio
EGD
- 4 - 3 - 2 - 1 0 1 2 3 4
atpC
atpD
atpG
atpA
atpH
atpF
atpE
atpB
atpI arcA
arcB
arcC
gadA
gadB
gadC
alsD
alsS
Log Ratio
FW04/0025 Acetoin biosynthesis
GAD system
ADI system
F-typeATPase
Responses to sodium diacetate stress more broadlysimilar between EGD and FW04/0025 – maindifferences focus in cell wall biogenesis
GENES FUNCTION EGD FW04/0025
Lip-1 cluster (lmo0201-0205)
virulence, intracellular survival 1.7, 5.2, 1.1, 1.9, 2.6 1.0, -1.4, -2.1, -1.8, 1.3
lmo0905 tyrosine protein phosphatase 2.9 1.4
lmo1301 putative acetyltransferase 3.1 1.1
lmo1460 (recO) DNA repair protein 2.5 1.4
lmo1508 two component histidine kinase 2.7 -1.5
lmo2677 uncharacterized hydrolase 8.5 -1.2
lmo2678-2682 (kdpEDCBA)
potassium transport (Kdp operon) 7.6, 10.1, 8.0, 7.9, 5.3 -1.2, 2.0, 1.3, 1.5, -2.2
EGD specifically upregulates K+ transport (kdp operon)and the Lip-1 cluster
Sodium diacetate specific genetic responses in EGD and FW04/0025
GENES REGULON FUNCTION EGD FW04/0025
FOLD CHANGES:CELL WALL/MEMBRANE RELATED:lmo0415 (pgdA) SigB peptidoglycan GlcNAc N-deacetylase -1.4 3.0lmo0582 (iap) SigB invasion protein (murein hydrolase) 1.2 2.6lmo0972-0974 (dltDCBA)
VirR, SigB, CodY
teichoic acid D-alanylation -1.5, 1.8, -1.8, 1.5 5.0, 5.9, 4.5, 5.5
lmo0995, lmo1291 putative peptidoglycan O-acetyltransferase -2.3, 1.4 3.9, 2.3
lmo2201-2202 (fabFH)
SigB fatty acid biosynthesis 1.4, -2.6 2.1, 7.4
lmo2504-2508 SigB murein hydrolases, cell division-associated proteins
1.2, -1.1, 1.3.1.1, 1.5 4.7, 2.4, 5.5, 2.1, 2.3
lmo2522 SigB, CodY unknown cell wall protein -4.9 9.5
CENTRAL METABOLISM:lmo1052-1055 (pdhABCD)
SigB pyruvate dehydrogenase 1.1, 1.3, 1.8, 2.2 5.7, 3.5, 5.5, 7.6
lmo1566 (citC) isocitrate dehydrogenase 1.3 3.3lmo2720 (acs) acetyl CoA synthetase 1.1 2.6
FW04/0025 specifically upregulates genes associated with the cell wall and aspects of central metabolism
Sodium diacetate specific genetic responses in EGD and FW04/0025
Exposure to sodium diacetate may lead tostrain specific responses relating to the cell wallPhysical lysis experiment testing cell wall “stability”
SD=sodium diacetate (20 mM)
terpenoidbackbones
Branched chainfatty acids
acetate
acetylphosphate
acetyl-CoApyruvatelactate
malonyl-CoA
acetoacetyl-CoAformate
oxaloacetate
TCA Cycle
2-acetolactate
acetoinBCAA
sugars
fatty acids
acs
ptb-buk-lpd-bkdA1A2-bkdB
alsD
alsS pta
lmo1381
pflA pflBC
ldh
pycA
pdhABCD
lmo1414
accABCD
ackA
lmo0722
ilvABCDHleuABCD
acetaldehyde
lmo1634
ethanol
A possible reason for FW04/0025 ability to resist sodium diacetate is that it can draw down on acetate poolse.g. synthesis of acetyl-CoA, lipids, acetoin
- 10 - 5 0 5 10
SigB(act)
PrfA(act)
HrcA(rep)
CtsR(rep)
VirR(act)
CodY(rep)
ns
ns
ns EGD
- 10 - 5 0 5 10
SigB(act)
PrfA(act)
HrcA(rep)
CtsR(rep)
VirR(act)
CodY(rep)
ns
ns FW04/0025
T-values T-values
Variations between strains seemed to be focussed in theCodY and VirR regulons as revealed using gene expression trend analysis (T-Profiler Boorsma et al.)
pH 5.0 vs pH 7.3Sodium diacetate(pH 5.0) vs pH 5.0
Bowman et al. Appl. Environ Microbiol submitted
Conclusions- Variation in resistance to acetate and sorbate associated with strains fromdifferent sources, possible influence of originating environment e.g. GI tract
- Acetate seems to augment tolerance to mineral acid
- Strain variation associated with aspects of the cell wall and central metabolism.Could affect diffsuion of unprotonated acetate and/or intracellular acetate pools
Future work- Need to do more proteomics to better define strain variation
- Cell wall chemistry alterations need to be determined
-Role of regulons (such as VirR) in organic acid resistance
Acknowledgements
Students:Kim Jye Lee ChangTerry PinfoldAnn Koshy
Esta HagesRolf Nilsson
Colleagues:Tom Ross Mark TamplinLyndal Mellefont Tom McMeekin