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![Page 1: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/1.jpg)
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
![Page 2: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/2.jpg)
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
![Page 3: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/3.jpg)
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?
![Page 4: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/4.jpg)
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
![Page 5: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/5.jpg)
Survival in raw milk cheese (pH 4.5, 15C, aw 0.93) varies between strains
0
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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)
![Page 6: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/6.jpg)
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
![Page 7: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/7.jpg)
- 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)
![Page 8: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/8.jpg)
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
![Page 9: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/9.jpg)
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)
![Page 10: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/10.jpg)
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
![Page 11: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/11.jpg)
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
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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
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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
![Page 14: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/14.jpg)
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
![Page 15: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/15.jpg)
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
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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+
![Page 16: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/16.jpg)
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:
![Page 17: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/17.jpg)
EGD responds strongly to HCl acidic stress (pH 5.0) compared to FW04/0025
![Page 18: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/18.jpg)
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
![Page 19: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/19.jpg)
Responses to sodium diacetate stress more broadlysimilar between EGD and FW04/0025 – maindifferences focus in cell wall biogenesis
![Page 20: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/20.jpg)
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
![Page 21: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/21.jpg)
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
![Page 22: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/22.jpg)
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)
![Page 23: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/23.jpg)
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
![Page 24: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/24.jpg)
- 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
![Page 25: Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural.](https://reader038.fdocuments.net/reader038/viewer/2022110206/56649cdc5503460f949a7522/html5/thumbnails/25.jpg)
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
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Acknowledgements
Students:Kim Jye Lee ChangTerry PinfoldAnn Koshy
Esta HagesRolf Nilsson
Colleagues:Tom Ross Mark TamplinLyndal Mellefont Tom McMeekin