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