1. Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acids John P. Bowman TIAR/School of Agricultural Science University of Tasmania Hobart, Tasmania Australia

2. Are more “hardier/problematic” strains of L. monocytogenes emerging? Vast majority inactivated but maybe some survive select resistant /robust strains? Source e.g. an animal Processing storage Survives in food product e.g. RTE Environmental dispersal/re-dispersal

3. Can “persistence” be defined and attributed to biological mechanisms L. monocytogenes “persistent” strains Biofilms (surface attachment) ? Differential responses to stress? Broader capacity to survive in food suppy chain environmental niches?

4. 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

5. Survival in raw milk cheese (pH 4.5, 15  C, a w 0.93) varies between strains Esta Hages (PhD Thesis, University of Tasmania) Strain (serotype): + FW03/0035 (4b) ○ ScottA (4b) □ LO28 (1/2a) × ATCC 19115 (4b) ∆ 70-1700 (4e)

6. End-products of L. monocytogenes metabolism of sugars – a common feature netween its biology and food preservation Aerobic Anaerobic

7. - 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 – does aciduricity relate to strain “success” (persistence)

8. Source: Genetic lineage/serotypeclinicalfood factory environmentcattlepoultrysheep other animals (goats, dogs, horses, wallabies) Lineage I:No. of strains: 1/2b1211 3b1 4b5192113 4e2121 Lineage II: 1/2a19337135 1/2c413 3a11 Lineage III: 4a21131 4c31 Strains examined for organic acid resistance – isolated from throughout the “food supply chain” Distribution of strain used to screen for relative organic acid resistance

9. Screened growth levels for 4 popular organic acid food 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)

10. Efect of isolation source on acetate and sorbate resistance Bowman et al. Appl. Environ Microbiol submitted Absolute net absorbance 21 mM sodium diacetate Absolute net absorbance poultry sheep cattle factory food clinical 23 mM potassium sorbate poultry sheep cattle factory food clinical

11. Absolute net absorbance 21 mM sodium diacetate 23 mM potassium sorbate Efect of genetic lineage of isolate on acetate and sorbate resistance

12. 0 SDA +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

13. Exponential phase/pH 5.0 (HCl) Stationary phase/pH 5.0 (HCl) FW04/0025 EGD The acid tolerance (pH 2.4, 2 hour exposure) of FW04/0025 was greater than EGD under all conditions except exponential phase at pH 7.3

14. 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 from both pH-dependent and phase-dependent adaptation

15. 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 SD BHI pH 5.0 +21 mM SD Sodium diacetate K+K+

16. 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) 5472108 45 26 116 12 6 6 2 4 5 1722 18 Bowman et al. Appl. Environ Microbiol submitted Number of genes differentially expressed:

17. EGD responds strongly to HCl acidic stress (pH 5.0) compared to FW04/0025

18. Responses of known pH homeostasis mechanisms in relation to different forms of acid stress: pH 5.0 – white bars pH 5.0 + 21 mM sodium diacetate – black bars Acetoin biosynthesis GAD system ADI system F-type ATPase

19. Responses to sodium diacetate stress more broadly similar between EGD and FW04/0025 – main differences focus in cell wall biogenesis

20. GENESFUNCTIONEGDFW04/0025 Lip-1 cluster (lmo0201-0205) virulence, intracellular survival1.7, 5.2, 1.1, 1.9, 2.61.0, -1.4, -2.1, -1.8, 1.3 lmo0905tyrosine protein phosphatase2.91.4 lmo1301putative acetyltransferase3.11.1 lmo1460 (recO)DNA repair protein2.51.4 lmo1508two component histidine kinase2.7-1.5 lmo2677uncharacterized hydrolase8.5-1.2 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

21. GENESREGULONFUNCTIONEGDFW04/0025 FOLD CHANGES: CELL WALL/MEMBRANE RELATED: lmo0415 (pgdA)SigBpeptidoglycan GlcNAc N-deacetylase-1.43.0 lmo0582 (iap)SigBinvasion protein (murein hydrolase)1.22.6 lmo0972-0974 (dltDCBA) VirR, SigB, CodY teichoic acid D-alanylation-1.5, 1.8, -1.8, 1.55.0, 5.9, 4.5, 5.5 lmo0995, lmo1291putative peptidoglycan O- acetyltransferase -2.3, 1.43.9, 2.3 lmo2201-2202 (fabFH) SigBfatty acid biosynthesis1.4, -2.62.1, 7.4 lmo2504-2508SigBmurein hydrolases, cell division- associated proteins 1.2, -1.1, 1.3.1.1, 1.54.7, 2.4, 5.5, 2.1, 2.3 lmo2522SigB, CodYunknown cell wall protein-4.99.5 CENTRAL METABOLISM: lmo1052-1055 (pdhABCD) SigBpyruvate dehydrogenase1.1, 1.3, 1.8, 2.25.7, 3.5, 5.5, 7.6 lmo1566 (citC)isocitrate dehydrogenase1.33.3 lmo2720 (acs)acetyl CoA synthetase1.12.6 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

22. Exposure to sodium diacetate may lead to strain specific responses relating to the cell wall Physical lysis experiment testing cell wall “stability” SD=sodium diacetate (20 mM)

23. A possible reason for FW04/0025 ability to resist sodium diacetate is that it can draw down on acetate pools e.g. synthesis of acetyl-CoA, lipids, acetoin

24. -10-505 SigB(act) PrfA(act) HrcA(rep) CtsR(rep) VirR(act) CodY(rep) ns EGD -10-505 SigB(act) PrfA(act) HrcA(rep) CtsR(rep) VirR(act) CodY(rep) ns FW04/0025 T-values Variations between strains seemed to be focussed in the CodY and VirR regulons as revealed using gene expression trend analysis (T-Profiler Boorsma et al.) pH 5.0 vs pH 7.3 Sodium diacetate(pH 5.0) vs pH 5.0 Bowman et al. Appl. Environ Microbiol submitted

25. Conclusions - Variation in resistance to acetate and sorbate associated with strains from different 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

26. Acknowledgements Students: Kim Jye Lee Chang Terry Pinfold Ann Koshy Esta Hages Rolf Nilsson Colleagues: Tom Ross Mark Tamplin Lyndal Mellefont Tom McMeekin