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Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acid

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.

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Phenotypic and corresponding transcriptomic responses of L. monocytogenes in the presence of unprotonated organic acid

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  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? Environmental dispersal/re-dispersal Processing storage Source e.g. an animal Survives in food product e.g. RTE Vast majority inactivated but maybe some survive select resistant /robust strains?

  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. stainless steel surface – different pH (24 h period) Sporadic strain Persistent strain L. monocytogenes biofilms and persistence pH 5.0 pH 7.3 pH 8.5 Nilsson et al Int. J. Food Microbiol. submitted

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

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

  7. Organic acids and L. monocytogenes – does aciduricity relate to strain “success” (persistence) • 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?

  8. 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) Potassium sorbate (3.3-33 mM) Potassium benzoate (2.3-23 mM) Sodium diacetate (0.35-70.3 mM)

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

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

  12. 0 SDA Growth rate is faster in resistant strain FW04/0025 compared to EGD but growth yield is the same when stressed with sodium diacetate +0 mM sodium diacetate +10 mM sodium diacetate +20 mM sodium diacetate Bowman et al. Appl. Environ Microbiol submitted

  13. 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 Exponential phase/pH 5.0 (HCl) Stationary phase/pH 5.0 (HCl) FW04/0025 FW04/0025 EGD EGD

  14. Sodium diacetate promotes acid tolerance arising from both pH-dependent and phase-dependent adaptation Exponential phase/pH 5.0 (HCl) + 21 mM sodium diacetate Stationary phase/pH 5.0 (HCl) + 21 mM sodium diacetate FW04/0025 FW04/0025 EGD EGD

  15. Resistant strains (FW04/0025, FW04/0023) accumulate less sodium diacetate and K+ compared to less resistant strains Sodium diacetate BHI pH 7.3 BHI pH 5.0 BHI pH 5.5 +21 mM SD BHI pH 5.0 +21 mM SD K+

  16. Microarray comparisons reveal several differences when strains are cultured in the presence of 21 mM sodium diacetate at pH 5.0 EGD (pH 5.0 + 21 mM SDA vs. pH 5.0) 6 45 2 4 6 5 26 18 17 22 116 12 FW04/0025 54 72 108 (pH 5.0 + 21 mM SDA vs. pH 5.0) EGD (pH 5.0 vs. pH 7.3) FW04/0025 (pH 5.0 vs. pH 7.3) Number of genes differentially expressed: Bowman et al. Appl. Environ Microbiol submitted

  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. EGD specifically upregulates K+ transport (kdp operon) and the Lip-1 cluster Sodium diacetate specific genetic responses in EGD and FW04/0025

  21. 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. 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.) FW04/0025 EGD ns ns CodY(rep) CodY(rep) VirR(act) VirR(act) ns ns CtsR(rep) CtsR(rep) HrcA(rep) HrcA(rep) ns PrfA(act) PrfA(act) SigB(act) SigB(act) - 10 - 5 0 5 10 - 10 - 5 0 5 10 T-values T-values Sodium diacetate(pH 5.0) vs pH 5.0 pH 5.0 vs pH 7.3 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

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