1. WORKPACKAGE 2.5: Potential risks associated with strategies DELIVERABLE 2.5.1: Data on the efficiency of current and new preservation/safety interventions against stress ‘hardened’ or adapted pathogens PILLAR 2: Control and intervention strategies along the fork-to-farm chain to ensure beef safety Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens Vienna, 25 - 26 March 2010

2. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 2 Aim of the study:  To investigate the potential effect of adaptive stationary phase ATR of L. monocytogenes cells on: (I) their attachment to SS under common food- relevant stresses (refrigeration, low pH, high salinity) & (II) the subsequent resistance of sessile cells to disinfection D2.5.1

3. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 3 Acid tolerance response (ATR) One of the most important and intensively investigated adaptive responses (Hill et al. 1995). Results from pre-exposure of bacterial cells to mild-acid conditions (pH 5.0 to 6.0) Salmonella enterica (Foster and Hall 1990) Escherichia coli O157:H7 (Leyer et al. 1995; Buchanan et al. 1999) L. monocytogenes (Gahan et al. 1996; O’Driscoll et al. 1996; Lou and Yousef 1996; 1997; Koutsoumanis et al. 2003; Koutsoumanis and Sofos 2004; Skandamis et al. 2008; 2009). D2.5.1

4. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 4 L. monocytogenes & ATR Besides enabling enhanced resistance to lethal acid exposure (pH ≈ 3) (Koutsoumanis et al. 2003; Koutsoumanis and Sofos 2004); it has also been found to offer cross-protection against: heat ethanol oxidative and osmotic stresses bacteriocin nisin and other ionophores (O’Driscoll et al. 1996; Datta and Benjamin 1997; Lou and Yousef 1997; Koutsoumanis et al. 2003; Skandamis et al. 2008; 2009) D2.5.1

5. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 5  Bacterial strain: Strain. Listeria monocytogenes Scott A (serotype 4b, epidemic strain, human isolate). TSB-G does not result in the formation of an acidic environment (lack of fermentable carbohydrate). TSB+G is acidogenic, supporting growth of the microorganism with an accompanying decrease of its pH.  Assay for ATR induction: After a preculture in TSB, bacteria were subcultured in either TSB+G or TSB-G (Buchanan and Edelson 1996). D2.5.1

6. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 6 Abiotic surface & bacterial attachment Stainless steel (SS) coupons (3 x 0.8 x 0.1 cm, type AISI-304). Setup: coupons were placed vertically in test tubes containing BHI broth Inoculation: ca. 10 8 CFU/ml of either nonadapted or acid-adapted cells. Incubation: statically for 10 d. D2.5.1

7. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 7 Cases examined for bacterial attachment 5 7.4 0.5 16 7.4 0.5 16 4.5 0.5 16 7.4 5.5 16 4.5 5.5 D2.5.1

8. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 8 Exposure of attached cells to disinfection SS coupon in BHI for 10 d SS coupon carrying attached bacteria REMOVALRINSINGDISINFECTION (6’) Attached L. monocytogenes cells to SS following acridine orange staining & epifluorescence microscopy observation D2.5.1

9. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 9 Exposure of attached cells to disinfection DISINFECTION (6’) HCL (pH 2) Lactic acid (pH 2) Essential oil of Satureja thymbra (1% v/v) Hydrosol of S. thymbra (100%) Satureja thymbra Essential oils & extracts of plants, herbs, and spices constitute a class of very potent natural antimicrobials (Skandamis and Nychas 2001; Nychas et al. 2003; Burt 2004). Essential oil and hydrosol of S. thymbra have recently been found to present sufficient bactericidal effect against monoculture and mixed-culture biofilms (Chorianopoulos et al. 2008). D2.5.1

10. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 10 DISINFECTION (6’) Quantification of viable attached bacterial population RINSING Quantification of sessile population Bead vortexing & agar plating Conductance measurements D2.5.1

11. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 11 Bead vortexing & agar plating Conductance measurements Vortexing (2’)Plate counting on TSA Incubation into Malthus module (30 o C for 48 h) SS coupon electrodes Conductance measuring tube Detection of attached cells by impedance measurements is already cited in the literature (Johnston and Jones 1995; Flint et al. 1997; Giaouris et al. 2005). D2.5.1 Quantification of viable attached bacterial population

12. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 12 Conductance measurements (MALTHUS) HOURS Detection time (DT) 05101520 1000 1500 2000 2500 3000 3500 4000 CONDUCTIVITY (μS) PC recording conductance changes Corresponding data [conductance (μS) vs time (h)] are recorded graphically as conductance growth curves. During bacterial growth, uncharged or weakly charged substrates (proteins, carbohydrates etc.) are converted into highly charged and products (amino acids etc.) Increase of broth conductance (Silley and Forsythe 1996) D2.5.1

13. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 13 inversely related to the initial bacterial population into the Malthus tubes  Longer DT suggests less remaining viable bacteria after disinfection (reduced resistance) D2.5.1 Conductance measurements (MALTHUS) HOURS Detection time (DT) 05101520 1000 1500 2000 2500 3000 3500 4000 CONDUCTIVITY (μS)

14. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 14 Results D2.5.1

15. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 15 Attachment of nonadapted ( ) & acid- adapted ( ) L. monocytogenes cells to SS ATR slightly increased attachment under mild acidic conditions, regardless of NaCl concentration. ≈ ≈ ≈ No effect when under natural pH conditions. D2.5.1

16. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 16 ATR & resistance of sessile cells to disinfection The effect of each disinfection treatment was expressed as the percentage of attached cells killed through the action of sanitizer: D2.5.1

17. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 17 Sanitation efficiency (%) : nonadapted : acid-adapted 5 o C, pH 7.4, 0.5% NaCl 16 o C, pH 7.4, 0.5% NaCl 16 o C, pH 4.5, 0.5% NaCl * Counts after disinfection below plate counting detection limit (1.03 log cfu/cm 2 ). A B C D2.5.1

18. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 18 : nonadapted : acid-adapted Sanitation efficiency (%) 16 o C, pH 4.5, 5.5% NaCl 16 o C, pH 7.4, 5.5% NaCl D E D2.5.1

19. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 19 For all studied cases, cells that had initially been acid adapted were more resistant to extreme acid challenge (pH 2), compared to nonadapted ones. Essential oil & hydrosol exhibited strong antimicrobial action, regardless of induction of ATR response. The mild acidic attachment conditions (pH 4.5) do not seem to confer any pH-inducible acid tolerance to L. monocytogenes cells that had not initially been acid adapted. Cells that had initially been acid adapted conserve their acid tolerance (even after 10 d under various environmental conditions). Acquired acid tolerance of L. monocytogenes was conserved even after several weeks of storage of the adapted bacteria at 4 o C (Phan-Thanh and Montagne 1998). In E. coli the acid resistance systems, once activated, remain active over prolonged periods of cold storage (Lin et al. 1996). Conclusions regarding ATR & disinfection D2.5.1

20. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 20 Bead vortexing & agar plating vs conductance method Conductance DTs corresponded well to agar plating results, since longer DTs correlated enough properly with higher sanitation efficiencies. D2.5.1

21. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Perspectives… Use a composite of strains to simulate better the conditions encountered in real complex food processing ecosystems. Different strains may respond differently to stresses and is likely that research investigating stress responses of only one strain may not always provide accurately evaluation of the worst-case scenario. Find and understand the molecular events probably jointly involved in L. monocytogenes stress adaptive responses and attachment to abiotic surfaces. D2.5.1

22. Laboratory of Microbiology & Biotechnology of Foods Agricultural University of Athens PILLAR 2, WP2.5, D2.5.6 Thank you Acknowledgments Dr Nikos Chorianopoulos Dr Panos Skandamis D2.5.1