1. Resistance – Thermal and Other Robert L. Buchanan DHHS Food and Drug Administration Center for Food Safety and Applied Nutrition

2. Thermal Resistance of E. sakazakii – Laboratory Trials Nazarowec-White and Farber (1997) Nazarowec-White and Farber (1997) Open stainless steel tubes in constant temperature water bath Open stainless steel tubes in constant temperature water bath 5 pooled food strains and 5 pooled clinical strains 5 pooled food strains and 5 pooled clinical strains Edelson-Mammel and Buchanan (2003) Edelson-Mammel and Buchanan (2003) Submerged coil apparatus Submerged coil apparatus 12 individual strains – food and clinical 12 individual strains – food and clinical

3. Thermal Resistance D-Value: D-Value: Time at a given temperature needed to reduce a microbial population by 90% Time at a given temperature needed to reduce a microbial population by 90% Z-Value: Z-Value: Change in temperature needed to change D-value by 90% Change in temperature needed to change D-value by 90%

4. Thermal Death Time Curves for 2 Enterobacter sakazakii Strains Heated at 58  C D = 30.5 sec D = 591.9 sec

5. Distribution of D 58°C -values for 12 Enterobacter sakazakii strains

6. Comparison of D 58°C -Values for Different Enterobacteriaceae

7. Effect of Heating Temperature on D- Value for E. sakazakii 607

8. Reported Z-values Nazarowec-White and Farber (1997) Nazarowec-White and Farber (1997) Pooled food isolates: 5.6°C Pooled food isolates: 5.6°C Pooled clinical isolates: 6.0 °C Pooled clinical isolates: 6.0 °C All: 5.8 °C All: 5.8 °C Edelson-Mammel and Buchanan (2003) Edelson-Mammel and Buchanan (2003) Strain 607: 5.6 °C Strain 607: 5.6 °C

9. Predicted Inactivation Results of these heating trials indicate that heating rehydrated infant formula at  70°C for even a few seconds will result in a substantial inactivation of E. sakazakii Results of these heating trials indicate that heating rehydrated infant formula at  70°C for even a few seconds will result in a substantial inactivation of E. sakazakii

10. Effect of Temperature of Water Used to Rehydrate Infant Formula Inoculated dried infant formula in baby bottle Add water pre-heated to different temperatures Cap and agitate periodically for 10 minutes Analyze rehydrated formula for E. sakazakii

11. Temperature Decline During Rehydration of Infant Formula

12. Rehydration of Dried Infant Formula Lower Limit of Detection

13. Impact of Heating on Nutrient Content Conducted by Atlanta Center for Nutrient Analysis (ACNA) Conducted by Atlanta Center for Nutrient Analysis (ACNA) Used boiling water as the worst case and compared against control Used boiling water as the worst case and compared against control Analyses done in triplicate Analyses done in triplicate Results expressed in terms of units/100 cal Results expressed in terms of units/100 cal

14. Impact of Heating on Nutrient Content Formula 1 Formula 2 Formula 3 Formula 4 Vitamin A 300/712686(-3.7)300/677678(+0.1)300/597613(+2.7)300/451459(+1.8) Vitamin D 60/7173(+2.8)60/5853(-8.7)60/5763(+10.8)60/5755(-2.3) Vitamin E 1.5/2.92.9(0.0)2.0/5.24.4(-14.0)1.4/3.13.0(-3.2)2.0/3.73.6(-3.5) Vitamin K 8.0/17.817.9(+0.6)8.0/9.49.7(+3.2)8.3/11.811.9(+0.9)8.2/15.414.8(-3.9) Thiamin100/223218(-2.2)80/168170(+1.2)100/164178(+8.5)60/141141(0.0)

15. Impact of Heating on Nutrient Content Formula 1 Formula 2 Formula 3 Formula 4 Riboflavin150/266261(-1.9)90/154143(-7.1)150/247257(+4.1)135/337308(+8.6) Vitamin B 6 60/8681(-6.4)60/114116(+1.8)62.5/136139(+2.2)75/118120(+1.7) Vitamin B 12 0.25/0.900.86(-4.4)0.30/0.720.72(0.0)0.30/0.400.41(+3.3)0.22/0.610.62(+2.0) Niacin1050/15031521(+1.2)1000/14181456(+2.7)750/12421236(-0.5)750/11761282(+9.0) Folic Acid 15.0/36.036.1(+1.0)16.0/32.333.3(+3.1)7.5/26.928.3(+5.2)15.0/43.945.2(+3.0)

16. Impact of Heating on Nutrient Content Formula 1 Formula 2 Formula 3 Formula 4 Pantothenic Acid 450/10101060(+5.0)500/793803(+1.3)450/633649(+2.5)450/770755(+1.9) Biotin4.4/9.910.4(+5.1)3.0/4.64.2(-8.0)5.5/6.66.7(+2.1)2.2/4.34.3(+1.9) Vitamin C 9.0/16.611.5(-30.7)12.0/26.29.0(-65.6)8.3/10.710.1(-5.6)8.0/18.315.0(-18.0)

17. Thermal Resistance of E. sakazakii – Pilot Plant Nazarowec-White et al. (1999) Nazarowec-White et al. (1999) Pooled strains? Pooled strains? HTST pasteurizer HTST pasteurizer

18. Effectiveness of Pasteurization 3 sec 10 sec 16 sec 30 sec 60 sec 59°C 100100 60 °C 6350 61 °C 100795020 62 °C 100403.23.2 63 °C 38.850.1200.630.40 64 °C 39.8165.00.0130.040 65 °C 31.62.00.200.0004 66 °C 6.310.200.079 67 °C 0.398 68 °C 0.063

19. Resistance to Other Treatments Great deal of information available about Enterobacteriaceae in general Great deal of information available about Enterobacteriaceae in general

20. Resistances If Like Other Enterobacteriaceae Not heat resistant Not heat resistant Moderate acid resistance if adapted Moderate acid resistance if adapted Moderate alkali resistance if adapted Moderate alkali resistance if adapted Low to moderate chlorine resistance Low to moderate chlorine resistance Low to moderate irradiation resistance Low to moderate irradiation resistance Will remain viable in refrigerated and frozen products for extended periods, particularly if neutral pH Will remain viable in refrigerated and frozen products for extended periods, particularly if neutral pH Moderate to good resistance to drying Moderate to good resistance to drying

21. Resistance to Other Treatments Little published information available specifically about E. sakazakii Little published information available specifically about E. sakazakii Even less available on distribution of resistances Even less available on distribution of resistances

22. Other Resistances Isolation from dried foods indicates resistance to drying Isolation from dried foods indicates resistance to drying Isolation from seeds treated with hypochlorite suggest that at least some strains may be relatively resistant to chlorine (Okuda et al., 1994) Isolation from seeds treated with hypochlorite suggest that at least some strains may be relatively resistant to chlorine (Okuda et al., 1994) Relatively sensitive to the growth inhibiting effects of chitosans (No et al., 2002) Relatively sensitive to the growth inhibiting effects of chitosans (No et al., 2002)

23. Resistance to Dehydration

24. Summary Not a particularly thermally resistant microorganism Not a particularly thermally resistant microorganism Substantial diversity in thermal resistance among strains Substantial diversity in thermal resistance among strains Good agreement among studies Good agreement among studies Inactivation at temperatures above 70°C, even for a few seconds Inactivation at temperatures above 70°C, even for a few seconds Specific information on its resistance to other treatments is generally lacking Specific information on its resistance to other treatments is generally lacking